Protein separation by electrophoresis

ABSTRACT

The present method provides methods and apparatus for separating proteins using a series of electrophoretic methods that utilize controlled fractionation and labeling techniques to resolve mixtures of proteins. The samples for each electrophoretic method other than the initial method, contain only a subset of proteins resolved in the preceding method. The methods can be used in a variety of different applications including, creating proteomic databases, comparative expression studies, diagnostics, structure activity relationships and metabolic engineering investigations.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisionalapplication 60/130,238 filed Apr. 20, 1999. This application is alsorelated to U.S. provisional application 60/075,715 filed Feb. 24, 1998,copending U.S. patent application Ser. No. ______, filed Feb. 25, 2000,entitled “Methods for Protein Sequencing,” and having attorney docketnumber 020444-000300US, and copending U.S. application Ser. No. ______,filed Feb. 25, 2000, entitled “Polypeptide Fingerprinting Methods andBioinformatics Database System,” and having attorney docket number020444-000100US. All of these applications are incorporated by referencein their entirety for all purposes.

FIELD OF THE INVENTION

[0002] This invention relates to the field of protein separation andproteomics.

BACKGROUND OF THE INVENTION

[0003] A goal of genomics research and differential gene expressionanalysis is to develop correlations between gene expression andparticular cellular states (e.g., disease states, particulardevelopmental stages, states resulting from exposure to certainenvironmental stimuli and states associated with therapeutictreatments). Such correlations have the potential to provide significantinsight into the mechanism of disease, cellular development anddifferentiation, as well as in the identification of new therapeutics,drug targets and/or disease markers. Correlations of patterns of geneexpression can also be used to provide similar insights into disease andorganism metabolism that can be used to speed the development ofagricultural products, transgenic species, and for metabolic engineeringof organisms to increase bioproduct yields or desirable metabolicactivities.

[0004] Many functional genomic studies focus on changes in mRNA levelsas being indicative of a cellular response to a particular condition orstate. Recent research, however, has demonstrated that often there is apoor correlation between gene expression as measured by mRNA levels andactual active gene product formed (i.e., protein encoded by the mRNA).This finding is not surprising since many factors-including differencesin translational efficiency, turnover rates, extracellular expression orcompartmentalization, and post-translational modification affect proteinlevels independently of transcriptional controls. Thus, the evidenceindicates that functional genomics is best accomplished by measuringactual protein levels (i.e., utilizing proteomic methods) rather thanwith nucleic acid based methods. The successful use of proteins forfunctional genomic analyses, however, requires reproduciblequantification of individual proteins expressed in cell or tissuesamples.

[0005] Two-dimensional (2-D) gel electrophoresis is currently the mostwidely adopted method for separating individual proteins isolated fromcell or tissue samples [5, 6, 7]. Evidence for this is seen in theproliferation (more than 20) of protein gel image databases, such as theProtein-Disease Database maintained by the NIH [8]. These databasesprovide images of reference 2-D gels to assist in the identification ofproteins in gels prepared from various tissues.

[0006] Capillary electrophoresis (CE) is a different type ofelectrophoresis, and involves resolving components in a mixture within acapillary to which an electric field is applied. The capillary used toconduct electrophoresis is filled with an electrolyte and a sampleintroduced into one end of the capillary using various methods such ashydrodynamic pressure, electroosmotically-induced flow, andelectrokinetic transport. The ends of the capillary are then placed incontact with an anode solution and a cathode solution and a voltageapplied across the capillary. Positively charged ions are attractedtowards the cathode, whereas negatively charged ions are attracted tothe anode. Species with the highest mobility travel the fastest throughthe capillary matrix. However, the order of elution of each species, andeven from which end of the capillary a species elutes, depends on itsapparent mobility. Apparent mobility is the sum of a specieselectrophoretic mobility in the electrophoretic matrix and the mobilityof the electrophoretic matrix itself relative to the capillary. Theelectrophoretic matrix may be mobilized by hydrodynamic pressuregradients across the capillary or by electroosmotically-induced flow(electroosmotic flow).

[0007] A number of different electrophoretic methods exist. Capillaryisoelectric focusing (CIEF) involves separating analytes (such asproteins) within a pH gradient according to the isoelectric point (i.e.,the pH at which the analyte has no net charge) of the analytes. A secondmethod, capillary zone electrophoresis (CZE) fractionates analytes onthe basis of their intrinsic charge-to-mass ratio. Capillary gelelectrophoresis (CGE) is designed to separate proteins according totheir molecular weight. (For reviews of electrophoresis generally, andCIEF and CZE specifically, see, e.g., Palmieri, R. and Nolan, J. A.,“Protein Capillary Electrophoresis: Theoretical and ExperimentalConsiderations for Methods Development,” in CRC Handbook of CapillaryElectrophoresis: A Practical Approach, CRC Press, chapter 13, pp.325-368 (1994); Kilar, F., “Isoelectric Focusing in Capillaries,” in CRCHandbook of Capillary Electrophoresis: A Practical Approach, CRC Press,chapter 4, pp. 95-109 (1994); and McCormick, R. M., “Capillary ZoneElectrophoresis of Peptides,” in CRC Handbook of CapillaryElectrophoresis: A Practical Approach, CRC Press, chapter 12, pp.287-323 (1994). All of these references are incorporated by reference intheir entirety for all purposes).

[0008] While 2-D gel electrophoresis is widely practiced, severallimitations restrict its utility in functional genomics research. First,because 2-D gels are limited to spatial resolution, it is difficult toresolve the large number of proteins that are expressed in the averagecell (1000 to 10,000 proteins). High abundance proteins can distortcarrier ampholyte gradients in capillary isoelectric focusingelectrophoresis and result in crowding in the gel matrix of size sievingelectrophoretic methods (e.g., the second dimension of 2-D gelelectrophoresis and CGE), thus causing irreproducibility in the spatialpattern of resolved proteins [20, 21 and 22]. High abundance proteinscan also precipitate in a gel and cause streaking of fractionatedproteins [20]. Variations in the crosslinking density and electric fieldstrength in cast gels can further distort the spatial pattern ofresolved proteins [23, 24]. Another problem is the inability to resolvelow abundance proteins neighboring high abundance proteins in a gelbecause of the high staining background and limited dynamic range of gelstaining and imaging techniques [25, 22]. Limitations with staining alsomake it difficult to obtain reproducible and quantifiable proteinconcentration values, with average standard variations in relativeprotein abundance between replicate 2-D gels reported to be 20% and ashigh as 45% [4]. In some recent experiments, for example, investigatorswere only able to match 62% of the spots formed on 3-7 gels run undersimilar conditions [21; see also 28, 29]. Additionally, many proteinsare not soluble in buffers compatible with acrylamide gels, or fail toenter the gel efficiently because of their high molecular weight [26,27].

SUMMARY OF THE INVENTION

[0009] The present invention provides a variety of electrophoreticmethods and apparatus for separating mixtures of proteins. The methodsinvolve conducting multiple capillary electrophoresis methods in series,wherein samples for each method other than the initial method containonly a subset of the proteins from the preceding step (e.g., fromfractions containing resolved protein from the preceding method). Byusing a variety of techniques to control elution during electrophoresis,the methods are capable of resolving proteins in even complex mixturessuch as obtained from tissues and native cells. Utilizing variouslabeling schemes and detection methods, certain methods can providequantitative information on the amount of each of the separatedproteins. Such information can be used in the development of proteindatabases in which proteins expressed under certain conditions arecharacterized and catalogued. Comparative studies to identify proteinsthat are differentially expressed between different types of cells ortissues can also be conducted with the methods of the present invention.The methods can also be used in diagnostic, structure activity andmetabolic engineering studies.

[0010] In general, the methods involve performing a plurality ofelectrophoretic methods in series. Each method in the series includeselectrophoresing a sample containing multiple proteins to obtain aplurality of resolved proteins. The sample that is electrophoresedcontains only a subset of the plurality of resolved proteins from theimmediately preceding method in the series (except the first method ofthe series in which the sample is the initial sample that contains allthe proteins). The resolved proteins from the final electrophoreticmethod are then detected using various techniques.

[0011] The electrophoretic methods typically are capillaryelectrophoresis methods, such as capillary isoelectric focusingelectrophoresis (CIEF), capillary zone electrophoresis (CZE) andcapillary gel electrophoresis (CGE), although the methods are amenableto other capillary electrophoresis methods as well. The particular orderof the methods can vary. Typically, the methods utilize combinations ofelectrophoretic methods which separate proteins on the basis ofdifferent characteristics (e.g., size, charge, isoelectric point).

[0012] In certain methods, the proteins are labeled to more easilydetect the resolved proteins, to alter the charge of the proteins, tofacilitate their separation, and/or to increase the signal-to-noiseratio. Labeling also enables certain methods to be conducted such thatthe resolved proteins obtained from the final electrophoretic method arequantitated. Quantitation allows the relative abundance of proteinswithin a sample, or within different samples, to be determined. Incertain methods, the time at which proteins are labeled is selected toprecede electrophoresis by capillary zone electrophoresis. Byselectively labeling certain residues, resolution of proteins duringcapillary zone electrophoresis can be increased.

[0013] Resolution, quantitation and reproducibility are enhanced byutilizing a variety of techniques to control elution of proteins duringan electrophoretic method. The particular elution technique employeddepends in part upon the particular electrophoretic method. However, ingeneral, hydrodynamic, salt mobilization, pH mobilization andelectroosmotic flow are utilized to controllably elute resolved proteinsat the end of each electrophoretic separation.

[0014] Some methods provide for additional analysis after theelectrophoretic separation. The type of analysis can vary and include,for example, infra-red spectroscopy, nuclear magnetic resonancespectroscopy, UV/VIS spectroscopy, fluorescence spectroscopy, andcomplete or partial sequencing. In certain methods, proteins in thefinal fractions are further analyzed by mass spectroscopy to determineat least a partial sequence for each of the resolved proteins (i.e., todetermine a protein sequence tag).

[0015] Thus, certain other methods involve performing one or morecapillary electrophoretic methods, each of the one or more methodsinvolving: (i) electrophoresing a sample containing multiple proteinswithin an electrophoretic medium contained within a capillary, and (ii)withdrawing and collecting multiple fractions, each fraction containingproteins resolved during the electrophoresing step. Each method in theseries is conducted with a sample from a fraction collected in thepreceding electrophoretic method, except the first electrophoreticmethod which is conducted with a sample containing the original mixtureof proteins. The proteins are labeled prior to conducting the lastelectrophoretic method. Either the proteins in the initial sample arelabeled (i.e., labeling precedes all the electrophoretic separations),or the proteins contained in fractions collected are labeled prior tothe last electrophoretic method. The final electrophoretic method isperformed, and resolved protein within, or withdrawn from, the capillaryutilized to conduct the final method is detected with a detector. Hence,the detector is adapted to detect resolved protein within the capillaryused in the final method or is connected in line with the capillary todetect resolved proteins as they elute from the capillary. In someinstances, the detected proteins are quantitated and further analyzed bymass spectroscopy to determine their relative abundance and/or toestablish a protein sequence tag for each resolved protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic representation of one example of anelectrophoretic system that can be utilized with certain methods of theinvention.

[0017]FIG. 2A is a schematic representation of some of the majorelements of an electrophoretic system utilized in conducting certainelectrophoretic methods of the invention.

[0018]FIG. 2B is a cross-sectional view of a capillary showing theorientation of a porous plug inserted into the capillary to controlelectroosmotic flow in certain methods of the invention.

[0019]FIGS. 3A and 3B are top-views of certain elements of microfluidicdevices that can be utilized to'conduct certain electrophoretic methodsof the invention.

[0020]FIG. 4 is an electropherogram for a sample containing fiveunlabeled proteins (hen egg white conalbumin, bovine serum albumin,bovine carbonic anhydrase II, carbonic anhydrase II, rabbit muscleGAPDH, and bovine ribonuclease A) as obtained following electrophoresisby capillary zone electrophoresis. Absorbance was monitored at 214 nm.Under the conditions of this particular experiment (see Example 1) inwhich the proteins were unlabeled, the proteins were not resolved.

[0021]FIG. 5 is a plot of electrophoretic mobility for each of the fiveproteins listed in FIG. 4 under the same electrophoresis conditions asdescribed in FIG. 4.

[0022]FIG. 6 is a plot showing the correlations between electrophoreticmobility and the predicted mass-to-charge ratio of the proteins at pH4.0.

[0023]FIG. 7 is an electropherogram obtained during separation of asample containing five sulfophenylisothiocyanate-labeled proteins (henegg white conalbumin, bovine serum albumin, bovine carbonic anhydraseII, carbonic anhydrase II, rabbit muscle GAPDH, and bovine ribonucleaseA) as obtained following electrophoresis by capillary zoneelectrophoresis. Absorbance was monitored at 214 nm. Under theconditions of this particular experiment (see Example 2) in which theproteins were labeled, the labeled proteins were partially resolved.

[0024]FIG. 8 is an electropherogram obtained during separation of asample containing the proteins hen white conalbumin, bovine serumalbumin, and bovine carbonic anhydrase II, by CIEF.

[0025]FIG. 9 is an electropherogram of a fraction (fraction F) obtainedfrom the separation by CIEF shown in FIG. 7.

[0026]FIG. 10 is an electropherogram of a fraction (fraction G) obtainedfrom the separation by CIEF shown in FIG. 7.

DETAILED DESCRIPTION

[0027] I. Overview

[0028] The present invention provides methods and apparatus forachieving the separation of proteins, including significant resolutionof proteins in complex mixtures from native cell and tissue samples. Theinvention is based in part upon the recognition that multidimensionalelectrophoretic methods involving multiple (typically different)electrophoretic methods performed in series utilizing controlledfractionation techniques to obtain defined fractions can be used toachieve high resolution of proteins. Labeling and detection steps can beincluded to increase sensitivity, alter the separation coordinates ofthe proteins, and to obtain accurate and reproducible quantitativeinformation about the resolved proteins. Typically, the electrophoreticmethods are capillary electrophoresis methods, particularly combinationsof capillary isoelectric focusing (CIEF), capillary zone electrophoresis(CZE) and capillary gel electrophoresis (CGE).

[0029] Several features enable methods to be performed in a controlledand reproducible fashion. For example, once proteins have had anopportunity to fractionate within the electrophoretic medium containedwithin a capillary, elution conditions are tailored so that separatedproteins are eluted in a controlled fashion to yield defined fractionsin which the proteins contained within a fraction fall within a certainpH range, electrophoretic mobility range, or molecular weight range, forexample. In certain methods, proteins are labeled at a selected stage ofthe separation process and the labeled proteins detected using adetector. Labeling enables proteins present at low concentration to moreeasily be detected and enhances reproducibility by increasingsignal-to-noise ratios. The detector can be used to detect proteins asseparated within an electrophoretic cavity or after they are eluted fromthe cavity. The combination of labeling and detection also enablesseparated proteins to be quantified. The combination of labeling andseparation can alter the net charge or solubility of the proteinscausing a change in their separation coordinates, for example, theirseparation order, the fraction in which they are collected, and elutiontime.

[0030] If additional information is desired, the methods can be expandedto include further analysis by techniques besides electrophoresis. Forexample, in certain methods, fractions collected from the finalelectrophoretic method are individually analyzed by mass spectroscopy toobtain additional information, such as molecular weight and partialsequence.

[0031] Quantitative detection and the ability to automate the methodsmeans that the methods are amenable to a variety of screening,comparative and diagnostic studies. For example, the methods can beutilized to develop comparative protein expression data. Suchcomparative studies can be utilized to identify markers of specificdiseases, potential targets for pharmaceuticals and/or drug candidates.Once markers that are selectively expressed in certain disease states,for example, are identified, the methods of the invention have utilityin diagnostic applications. The methods of the invention can also beutilized to develop a protein database that includes, for example,separation coordinates, isoelectric points, apparent molecular weightsand relative abundance information for proteins in different cells,tissues or states. The methods also find utility in studies onstructure/activity relationships and in metabolic engineeringinvestigations in which one genetically modifies a certain gene and thendetermines what effects such a modification has on cellular proteinexpression.

[0032] II. Separation Methods

[0033] A. General

[0034] The methods of the present invention utilize a combination ofelectrophoretic methods conducted in series to resolve mixtures ofproteins. The methods are said to be conducted in series because thesample(s) electrophoresed in each method are from solutions or fractionscontaining proteins electrophoresed in the preceding method, with theexception of the sample electrophoresed in the initial electrophoreticmethod. As used herein, the terms protein, peptide and polypeptide areused interchangeably and refer to a polymer of amino acid residues. Theterm also applies to amino acid polymers in which one or more aminoacids are chemical analogues of corresponding naturally-occurring aminoacids, including amino acids which are modified by post-translationalprocesses (e.g., glycosylation and phosphorylation).

[0035] The series of electrophoretic methods are typically conducted insuch a way that proteins in an applied sample for each electrophoreticmethod of the series are isolated or resolved physically, temporally orspacially to form a plurality of fractions each of which include only asubset of proteins of the applied sample. Thus, a fraction refers to aprotein or mixture of proteins that are resolved physically, temporallyor spacially from other proteins in a sample subjected toelectrophoresis. Resolved proteins can refer to a single species or amixture of proteins that are separated from other proteins during anelectrophoretic method. As just noted, samples in the variouselectrophoretic methods are obtained from such fractions, with theexception of the first electrophoretic method in which the sample is theoriginal sample containing all the proteins to be separated.

[0036] Typically, these multiple electrophoretic methods in the seriesseparate proteins according to different characteristics. For example,one method can separate proteins on the basis of isoelectric points(e.g., capillary isoelectric focusing electrophoresis), other methodscan separate proteins on the basis of their intrinsic or induced(through the application of a label to certain ionizable amino acidresidues) charge-to-mass ratio at any given pH (e.g., capillary zoneelectrophoresis), whereas other methods separate according to the sizeof the proteins (e.g., capillary gel electrophoresis). Such approachesthat separate proteins through a series of electrophoretic methods arereferred to herein as “multidimensional” electrophoretic methods,wherein each particular electrophoretic method constitutes a“dimension.”

[0037] Apparatus used to conduct various electrophoretic methods areknown in the art. In general, however, and as shown in FIG. 2A, thebasic configuration of a typical capillary electrophoretic systemutilized in certain methods of the invention includes a capillary 8having two ends 10, 12. One end 10 is in contact with an anode solutionor anolyte 14 contained in an anode reservoir 18 and the other end 12 isin contact with a cathode solution or catholyte 16 in a cathodereservoir 20. One electrode (the anode) 22 is positioned to be inelectrical communication with the anode solution 14 and a secondelectrode 24 is positioned to be in electrical communication with thecathode solution 16. The cavity 26 of the capillary 8 is filled with anelectrophoretic medium, which in some instances can include a polymermatrix. As used herein, the term anode refers to the positively chargedelectrode. Thus, negatively charged species move through theelectrophoretic medium toward the anode. The term cathode refers to thenegatively charged electrode; positively charged species migrate towardthis electrode. The anolyte is the solution in which the anode isimmersed and the catholyte is the solution in which the cathode isimmersed.

[0038] Sample is introduced into the capillary 8 via an inlet 28, andthe protein components therein resolved as an electrical field isapplied between the two electrodes 22, 24 by a power source 32 and theproteins separate within the electrophoretic medium contained within theseparation cavity 26. Protein components can be controllably eluted fromthe capillary via outlet 30 by controlling various parameters such aselectroosmotic flow (see infra) and/or by changing the composition ofone or both of the reservoir solutions (e.g., adjusting the pH or saltconcentration). Typically, the inlet 28 and the outlet 30 are simplyportions of the capillary formed to allow facile insertion into acontainer containing sample, anolyte or catholyte.

[0039] The term “capillary” as used in reference to the electrophoreticdevice in which electrophoresis is carried out in the methods of theinvention is used for the sake of convenience. The term should not beconstrued to limit the particular shape of the cavity or device in whichelectrophoresis is conducted. In particular, the cavity need not becylindrical in shape. The term “capillary” as used herein with regard toany electrophoretic method includes other shapes wherein the internaldimensions between at least one set of opposing faces are approximately2 to 1000 microns, and more typically 25 to 250 microns. An example of anon-tubular arrangement that can be used in certain methods of theinvention is the a Hele-Shaw flow cell [67, 68]. Further, the capillaryneed not be linear; in some instances, the capillary is wound into aspiral configuration, for example.

[0040] An example of a system utilized with certain methods of theinvention is illustrated in FIG. 1. This particular example shows asystem in which three electrophoresis methods (initial, intermediate andfinal methods) are linked. The particular number of electrophoreticmethods conducted can vary, although the methods of the inventioninclude at least two electrophoretic methods. Most typically, themethods utilize two or three electrophoretic separation methods.

[0041] As can be seen in FIG. 1, an initial sample containing aplurality of proteins is introduced from sample container 50 into afirst separation cavity of a first capillary 54 via sample inlet 52utilizing any of a number of methods known in the art. Examples ofsuitable methods include, pulling sample into the sample inlet 52 undervacuum (e.g., by pulling a vacuum on the sample outlet) or pushingsample into the sample inlet 52 by pressurizing the sample container 50.Electromigration, often referred to as electrokinetic injection, isanother option. Once the initial sample is introduced into sample inlet52, the sample is then electrophoresed within the first separationcavity within the first capillary 54. The first separation cavitycontains a desired electrophoretic medium in which proteins in theinitial sample are at least partially resolved. Electrophoretic mediumcontaining resolved proteins is withdrawn from the first cavity,typically out the end of the separation cavity opposite the end in whichsample was introduced, although other withdrawal sites can be utilized(see infra). The withdrawn medium travels through outlet 56 and iscollected in separate containers 58 as multiple fractions. As shown inFIG. 1B, the containers 58 into which fractions are collected aretypically associated with a fraction collection device (a portion ofwhich is shown 60) capable of automatically advancing a set ofcontainers 58 to collect defined fractions (e.g., fractions of a certainvolume or covering a selected pH range).

[0042] A sample from a fraction collected from the first electrophoreticmethod is then withdrawn from one of the plurality of containers 58,again utilizing techniques such as those described supra, via a secondsample inlet 62. Proteins in the sample from the fraction can then befurther resolved by conducting an intermediate electrophoretic method(in the example shown in FIG. 1, the second electrophoretic method). Thesample is introduced into a second capillary 64 via inlet 62 and theproteins within the sample further separated within the electrophoreticmedium contained within the second separation cavity of the secondcapillary 64 and then eluted from the cavity via outlet 66. As with thefirst electrophoretic separation, the electrophoretic medium containingthe resolved or partially resolved proteins is collected as separatefractions within containers 68 typically aligned and advanced by asecond fraction collection device (a portion of which is shown 70).

[0043] A process similar to the second/intermediate method is conductedduring the final electrophoretic method (the third electrophoreticseparation method shown in FIG. 1). Sample is drawn via inlet 72 from acontainer 68 containing a fraction obtained during the preceding methodand is introduced into a third or final electrophoretic cavity of athird capillary 74 containing a third electrophoretic medium in whichproteins contained in the applied sample are separated still further yetby electrophoresis. The third electrophoretic medium containing thefurther isolated proteins is subsequently withdrawn through outlet 76.

[0044] As noted above, more than the three electrophoretic methods shownin FIG. 1 can be performed. Such methods essentially involve repeatingthe general steps described for the second/intermediate electrophoreticseparation above one or more times.

[0045] Following the final electrophoretic separation, a variety ofdifferent options for analyzing the resolved proteins are available. Asshown in FIG. 1, withdrawn electrophoretic medium can be passed througha detector 78 in fluid communication with the separation cavity of thelast capillary 74 to detect the resolved proteins. The detector 78, oran optional quantifying device capable of receiving a signal from thedetector (not shown), can be used to quantitate the amount of proteinwithin a certain portion or fraction of the electrophoretic medium.

[0046] Alternatively, or in addition, fractions can be taken from theelectrophoretic medium exiting the final capillary 74 or the detector 78and analyzed by an analyzer 82 using some technique other thanelectrophoresis. Examples of such techniques include variousspectroscopic methods (e.g., IR, UV/VIS and NMR) and various massspectroscopy methods (e.g., electrospray ionization-time of flight[ESI-TOF] mass spectroscopy). Mass spectral data, for example, can beutilized to deduce a partial or full sequence of the protein(s) (i.e.,determine a protein sequence tag) within a particular fraction. FIG. 1depicts a situation in which sample is withdrawn via line 80 (dashed toindicate optional nature of this step) to another analyzer 82 (e.g.,mass spectrometer).

[0047] A number of other configurations can be utilized. For example,the capillaries and detector(s) can be fabricated within a microfluidicchip (see infra).

[0048] The specific elution conditions utilized to withdraw resolvedproteins from the separation cavity depends upon the type ofelectrophoretic method conducted and is described more fully below foreach of the electrophoretic methods typically utilized in the presentinvention. In general, however, once proteins have been resolved, theconditions within the separation cavity are adjusted as necessary (orthe initial conditions selected) to achieve selective or controlledelution of the proteins from the cavity. For example, elution can beachieved by adding salts to, or adjusting the pH of, the anode orcathode solution, by regulating electroosmotic flow, by applyinghydrodynamic pressure or combinations of the foregoing.

[0049] Using the methods of the invention, resolved proteins can beisolated physically (e.g., placement into different containers such asillustrated in FIG. 1), spatially (e.g., spread throughout theelectrophoretic medium contained in the separation cavity) and/ortemporally (e.g., controlling elution so different proteins within asample elute from the capillary at different times). Thus, the methodsof the invention can separate mixtures of proteins as a function of thecomposition of elution buffers and/or time, and are not limited to thespatial separation of proteins as are certain traditionaltwo-dimensional (2-D) gel electrophoresis systems. Instead, withcontrolled elution, fractions can be collected so that proteins within afraction fall within a range of isoelectric, electrophoretic mobility,or molecular weight values, for example. Controlled elution of proteinsmeans that methods can be performed in a reproducible fashion. Suchreproducibility is important in conducting comparative studies and indiagnostic applications, for example.

[0050] During the elution or withdrawing of resolved proteins, generallyonly a portion of the electrophoretic medium containing the resolvedproteins is typically collected in any given fraction. This contrastswith certain 2-D methods in which a gel containing all the resolvedproteins is exuded from the separation cavity and the exuded gelcontaining all the proteins is used to conduct another electrophoreticseparation.

[0051] Spacially, physically or temporally resolved proteins obtained atthe conclusion of one electrophoretic method are then used as the sourceof samples for further separation of proteins contained within thefraction during a subsequent electrophoretic method. As illustrated inFIG. 1, typically samples from different resolved fractions aresequentially electrophoresed on the same capillary. Normally anothersample is not applied until the proteins in the preceding sample aresufficiently withdrawn from the separation cavity so that there is nooverlap of proteins contained in different fractions. Sequential elutionof fractions through the same column can significantly reduce oreliminate variations resulting from differences in cross-linking orelectric field strength that can be problematic in certain slab gelelectrophoretic methods. Hence, sequential separation can furtherenhance the reproducibility of the methods of the invention. Othermethods, however, can be performed in a parallel format, wherein samplesfrom different fractions are electrophoresed on separate capillaries.This approach allows for separations to be completed more quickly.However, the use of multiple capillaries can increase the variability inseparation conditions, thereby reducing to some extent reproducibilitybetween different samples.

[0052] In certain methods, proteins are labeled at a selected stage ofthe separation process and then detected using the detector. Labelingenables proteins present at low concentration to more easily be detectedand enhances reproducibility by increasing signal-to-noise ratios. Thedetector can be used to detect proteins as separated within anelectrophoretic cavity or after they are eluted from the cavity. Thecombination of labeling and detection also enables separated proteins tobe quantified. The point in the overall method at which labeling isconducted depends in part on the particular electrophoretic methodsbeing conducted as discussed more fully below. In general, however,labeling is typically conducted before a gel capillary electrophoreticseparation is performed; whereas, labeling is normally conducted aftercapillary isoelectric focusing is performed rather than before. Labelingcan also be used before a zone capillary electrophoresis separation isperformed as a means to modify the net charge on the proteins and theirrelative electrophoretic mobilities.

[0053] As noted above, some of the more commonly used electrophoreticmethods utilized in the present invention are capillary isoelectricfocusing electrophoresis, capillary zone electrophoresis and capillarygel electrophoresis. Specific issues regarding the performance of thesemethods are described in the following sections.

[0054] B. Capillary Isoelectric Focusing Electrophoresis (CIEF)

[0055] 1. General

[0056] Isoelectric focusing is an electrophoretic method in whichzwitterionic substances such as proteins are separated on the basis oftheir isoelectric points (pI). The pI is the pH at which a zwitterionicspecies such as a protein has no net charge and therefore does not movewhen subjected to an electric field. In the present invention, proteinscan be separated within a pH gradient generated using ampholytes orother amphoteric substances within an electric field. A cathode islocated at the high pH side of the gradient and an anode is located atthe low pH side of the gradient. Proteins introduced into the gradientfocus within the pH gradient according to their isoelectric points andthen remain there. General methods for conducting CIEF are described,for example, by Kilar, F., “Isoelectric Focusing in Capillaries,” in CRCHandbook on Capillary Electrophoresis: A Practical Approach, CRC Press,Inc., chapter 4, pp. 95-109 (1994); and Schwartz, H., and T. Pritchett,“Separation of Proteins and Peptides by Capillary Electrophoresis:Application to Analytical Biotechnology,” Part No. 266923(Beckman-Coulter, Fullerton, Calif., 1994); Wehr, T., Rodriquez-Diaz,R., and Zhu, M., “Capillary Electrophoresis of Proteins,” (MarcelDekker, NY, 1999), which are incorporated herein by reference in theirentirety.

[0057] 2. System and Solutions

[0058] Because CIEF is primarily an equilibrium technique with lowcurrent densities, capillary heating typically is not a problem.Therefore, fairly large bore capillaries can be utilized. Suitable sizesinclude, but are not limited to, capillaries having internal diametersof 2-600 μm, although more typically capillaries having internaldiameters of 25-250 μm are utilized. The use of relatively large borecapillaries means the method can use relatively high protein loads,which facilitates detection in the following dimension(s). This featureof CIEF makes the method well-suited for the initial or one of the earlyelectrophoretic separations in the series. However, smaller diametercapillaries enable temperature to be controlled more carefully and, insome methods, result in improved signal detection (e.g., by laserinduced fluorescence (LIF) detection of fluorescently labeled proteins).

[0059] The capillaries can have varying lengths. The length selecteddepends in part on factors such as the extent of separation required.Typically, the capillaries are about 10 to 100 cm in length, althoughsomewhat shorter and longer capillaries can be used. While longercapillaries typically result in better separations and improvedresolution of protein mixtures, longer capillaries also afford moreopportunities for protein-wall interactions and lower field strength.Consequently, there tends to be an upper limit on capillary lengthbeyond which resolution may be lost. Longer capillaries can be ofparticular use in resolving low abundance proteins. Further guidance onsize and length of capillaries is set forth, for example, in Palmieri,R. and J. A. Nolan, “Protein capillary electrophoresis: Theoretical andexperimental considerations for methods development,” in: CRC Handbookof Capillary Electrophoresis: A Practical Approach, Chp. 13, pgs.325-368 (CRC Press, Boca Raton, 1994).

[0060] Generally, the capillaries are composed of flised silica,although plastic capillaries and PYREX (i.e., amorphous glass) can beutilized in certain methods. As noted above, the capillaries do not needto have a round or tubular shape. Other shapes wherein the internaldimension between opposing faces is within the general range set forthin this section can also be utilized.

[0061] A variety of different anode and cathode solutions can be used.Common solutions include sodium hydroxide as the catholyte andphosphoric acid as the anolyte. Similarly, a number of differentampholytes can be utilized to generate the pH gradient, includingnumerous commercially available ampholyte solutions (e.g., BioLyte,Pharmalyte and Servalyte). The selection of ampholytes and the breadthof the ampholyte gradient can impact the resolution that is achieved byCIEF methods. Narrow ampholyte gradients increase the number oftheoretical plates in the separation and can be beneficial for higherresolution-separations over narrow pI ranges.

[0062] CIEF methods utilized in the separations of the invention can beconducted in capillaries containing polymeric matrices or in freesolution (i.e., no gel or other polymeric matrix). Polymer matrices aretypically added to slow electroosmotic flow; however, in some instances,inclusion of polymeric matrices can restrict movement of larger proteins(see, e.g., Patton, 26). The use of free solutions is preferable in suchcases possibly in combination with other methods (e.g., capillarycoatings, gel plugs, or induced electric fields) to control theelectroosmotic flow.

[0063] 3. Sample Preparation

[0064] Typically protein samples to be electrophoresed by CIEF aredenatured prior to loading the sample into the capillary. This ensuresthat the same proteins all have the same charge and thus identicalproteins focus at the same location rather than potentially at multiplezones within the capillary. Denaturants (e.g., urea), non- andzwitterionic-surfactants (e.g., IGEPAL CA-630 or3-[{3-cholamidopropyl}dimethylammonio]-1-propane sulfonate) can also beused to suppress protein-wall and/or protein-protein interactions thatcan result in protein precipitation. Another advantage of denaturing theproteins prior to electrophoresis is that the results can be used incomparisons with archival data typically obtained under denaturingconditions.

[0065] A typical denaturing buffer includes urea and a nonionic orzwitterionic surfactant as denaturants; a reducing agent (e.g.,dithiothreitol (DTT) or mercaptoethanol) is typically included to reduceany disulfide bonds present in the proteins. Other denaturants besidesurea that can be used include, but are not limited to, thiourea anddimethylformamide (DMF). Generally, guanidine hydrochloride is notutilized as a denaturant because of the very high ionic strength itimparts to a sample. Exemplary neutral detergents includepolyoxyethylene ethers (“tritons”), such as nonaethylene glycoloctylcyclohexyl ether (“TRITON” X-100), polyglycol ethers, particularlypolyalkylene alkyl phenyl ethers, such as nonaethylene glycoloctylphenyl ether (“NONIDET” P-40 or IGEPAL CA-630), polyoxyethylenesorbitan esters, such as polyoxyethylene sorbitan monolaurate(“TWEEN”-20), polyoxyethylene ethers, such as polyoxyethylene laurylether (C₁₂E₂₃) (“BRIJ”-35), polyoxyethylene esters, such as 21 stearylether (C₁₈E₂₃) (“BRIJ”721), N,N-bis[3-gluconamido-propyl]cholamide(“BIGCHAP”), decanoyl-N-methylglucamide, glucosides such asoctylglucoside, 3-[{3-cholamidopropyl}dimethylammonio]-1-propanesulfonate and the like.

[0066] The optimal amount of denaturant and detergent depends on theparticular detergent used. In general the denaturing sample bufferscontain up to 10 M urea (more typically 4-8 M and most typically 6-8 M).Specific examples of suitable buffers (and denaturants and nonionicsurfactants for inclusion therein) include those described byHochstrasser et. al.[5] and O'Farrell[6]. Denaturation is typicallyadvanced by heating for 10 min at 95° C. prior to injection into thecapillary. Adjustments in the denaturing sample buffers are made asnecessary to account for any electroosmotic flow or heating effects thatoccur (see, e.g., Kilar, F., “Isoelectric Focusing in Capillaries,” inCRC Handbook on Capillary Electrophoresis: A Practical Approach, CRCPress, Inc., chapter 4, pp. 95-109 (1994)).

[0067] The amount of protein within a sample can vary and, as notedabove, depends in part of the size of the capillary used. In general,the capillary is loaded with 0.1 to 5.0 mg of total protein. Samples canbe spiked with one or more known pI standards to assess the performanceof the method.

[0068] 4. Elution

[0069] A variety of techniques can be utilized to elute or withdrawelectrophoretic medium containing resolved proteins out from thecapillary, but these methods fall into three general categories:hydrodynamic elution, electroelution and control of electroosmotic flow.

[0070] a. Hydrodynamic/Pressure Elution

[0071] Hydrodynamic or pressure elution involves applying pressure (orpulling a vacuum) via an appropriate pump connected with one end of thecapillary (see, e.g. Kilar, F., “Isoelectric Focusing in Capillaries,”in CRC Handbook on Capillary Electrophoresis: A Practical Approach, CRCPress, Inc., chapter 4, pp. 95-109 (1994)). However, hydrodynamicelution can cause band broadening and loss of resolution due to theparabolic flow profile that is formed in the capillary.

[0072] b. Electroelution

[0073] Electroelution, the other major approach, encompasses a varietyof techniques and in general involves altering the solution at the anodeand/or cathode to change some parameter (e.g., pH, ionic strength, saltconcentration) of the electrophoretic medium in the separation cavitysufficiently to effect elution.

[0074] i. Salt mobilization

[0075] One electroelution approach involves addition of a salt to thecatholyte or anolyte, the salt having a non-acidic or non-basiccounterion of the same charge as the acidic or basic species within thereservoir to which the salt is added so that the counterion migratesfrom the reservoir into the capillary. Since electrical neutrality mustbe maintained within the capillary, the movement of the counterion intothe capillary results in a reduction of the concentration of protons orhydroxide within the capillary, and thus the pH is either raised orlowered. The theoretical basis for this type of mobilization isdescribed by S. Hjerten, J.-L. Liao, and K. Yao, J. Chromatogr., 387:127 (1987). For example, if the catholyte is sodium hydroxide (i.e., thebasic species is hydroxide) then a salt having a negatively chargedcounterion other than hydroxide is added, for example sodium chloride.Movement of chloride ion into the capillary reduces the localconcentration of hydroxide within the capillary, thereby decreasing thepH. As another example, if the anolyte is phosphoric acid, then a salthaving a counterion other than a proton is added, for example sodiumphosphate. In this instance, movement of sodium ion into the capillaryreduces the local concentration of protons within the capillary therebyincreasing the pH. As the pH is lowered or raised within regions of thecapillary due to the presence of the added counterion, elution occurssince the ampholytes, and the focused proteins, migrate to thenewly-defined pH regions corresponding to their isoelectric points. Ithas been shown that both the type and concentration of salt used formobilization has impact on the resolution of eluted protein peaks [R.Rodriguez-Diaz, M. Zhu, and T. Wehr, J. Chromatogr. A, 772:145 (1997)].In particular, the addition of sodium tetraborate instead of sodiumchloride to the catholyte results in greatly increased resolution ofseparated proteins.

[0076] ii. pH Mobilization

[0077] Another technique, referred to herein as “pH mobilization” canalso be utilized to elute proteins during CIEF. In this' approach, anadditive is added to either the anode or cathode solution to alter thepH of the solution. Unlike salt mobilization, however, the additive doesnot contribute a mobile counteribn that moves into the capillary. Here,the elution occurs as a result of the pH gradient being redefined by thepH of one or both of the reservoirs; therefore, proteins with pI's thatfall outside of this redefined pH gradient are eluted into either theanode or cathode reservoirs. Typically, the technique for cathodicmobilization would proceed as follows. Once the proteins are focused inan exemplary pI range of 3-10 using phosphoric acid as the anolyte andsodium hydroxide as the catholyte, the cathodic capillary end isimmersed into a reservoir containing a solution that has a pH slightlyless than 10, for example 50 mM imidazole (pKa 7) which has a pH of9.85. The proteins are then allowed to refocus in the capillary,recognizable by a stabilization of the current through the capillary,the pI range now being defined by 3-9.85. Any proteins with anisoelectric point of 9.85 to 10 are eluted into the catholyte. Theprocess can be repeated with catholyte containing a species that reducesthe pH to slightly less than 9.85. In a stepwise fashion, the pH can becontinued to be reduced to pH 7, thereby collecting separated proteinsin fractions that span the range of 7-10. At this point, anodicmobilization can proceed by replacing the anolyte with acids ofincreasing pKa to selectively increase the pH from 3 to 7, therebycollecting fractions in the acidic range (pH 3-7). The number offractions can vary depending on the desired fractionation resolution.Typically, these fractions are defined by differences of 0.05-0.5 pHunits.

[0078] The technique of pH mobilization can be useful for proteinsamples containing a high concentration of one or more proteins that maycause uneven spatial gradients inside the capillary. Using pHmobilization, only those proteins with isoelectric points below or abovethe pI range that is defined by the reservoir pH's are eluted. Thiselution would, therefore, be reproducible regardless of differences inthe shape of the capillary pH gradient or the presence of uneven spatialgradients inside the capillary.

[0079] c. Electroosmotic Flow (EOF)

[0080] Regulating the magnitude of electroosmotic flow (EOF)significantly affects the preceding electroelution methods (see supra)and is another means by which resolved proteins can be selectivelywithdrawn upon conclusion of an isoelectric focusing separation. EOF isgenerated by the ionization of silanol functionalities on the surface ofa silica capillary. Such ionization results in a layer of protons in theelectrophoretic medium at the surface of the silica capillary. Once anelectric field is applied, the layer of protons essentially constitutesa positively charged column of fluid which migrates toward the cathode,thereby causing bulk flow of the electrophoretic medium within thecapillary. Apparent velocity of analytes is equal to the sum of theelectroosmotic flow and their electrophoretic mobility. Thus, bycontrolling EOF, one can control or regulate the rate at which proteinsmove through the capillary. In CIEF methods, generally EOF should becontrolled to allow proteins within an injected sample sufficient timeto focus before the proteins begin eluting from the capillary.

[0081] A variety of techniques can be utilized to regulate EOF. Oneapproach involves coating the walls of capillaries with various agents.For example, EOF along glass silicate surfaces can be substantiallyreduced by silanizing them with a neutral silane reagent that masks asubstantial percentage of surface silanol groups (e.g., polyacrylamide,polyethylene glycol and polyethylene oxide). The magnitude of EOF can befurther controlled by using silanizing reagents that include positivelyr negatively charged groups. Positively charged coatings can be used tonullify surface negative charges to give a net surface charge of zero,so that EOF approaches zero. Coatings with higher positive chargedensities can be used to reverse the direction of EOF for chargedsurface materials. This can be useful for slowing the net migrationrates of positively charged sample species. Conversely, negativelycharged coatings can be used to impart to or increase the magnitude ofthe negative charge on surfaces, so as to increase the net migrationrates of negatively charged species. Representative positively chargedcoatings include trialkoxysilanes with polyethyleneimine, quaternizedpolyethyleneimine, poly(N-ethylaminoacrylamide) and chitosans, forexample. Representative negatively charged coatings includetrialkoxysilanes with carboxylate and sulfonate containing materialssuch as poly(methylglutamate) and 2-acrylamido-2-methylpropanesulfonatepolymers, for example. It will be recognized that charged coatings canalso effectively reduce sample adsorption, especially for samples havingthe same charge polarity as the coating.

[0082] The separation medium can also include soluble agents fordynamically coating the walls of the separation cavity, to help reduceEOF during electrophoresis. Such soluble coating agents includequaternary ammonium-containing polymers, methyl cellulose derivatives,cellulose acetate, polyethylene oxide, chitosan, polyvinyl alcohol,polyethylene glycol, polyethylenimine, and polyethyleneoxide-polypropylene oxide-polyethylene oxide triblock copolymers, forexample. Typically, soluble coating agents are included atconcentrations of about 0.05% to about 4%, and more typically of about1% to about 2%.

[0083] EOF and sample absorption can also be adjusted by includingsuitable reagents in the separation medium and running buffers. Forexample, negative surface charges can be masked by including a cationicadditive in the medium, such as metal amine complexes, amines andpolyamines such as propylamine, triethylamine, tripropylamine,triethanolamine, putrescine, spermine, 1,3-diaminopropane, morpholine,and the like. Zwitterionic species comprising both negatively andpositively charged groups that are isoelectric at the pH ofelectrophoresis can also be used, such as trialkylammonium propylsulfonates, where alkyl is methyl, ethyl, propyl, and longer alkylchains.

[0084] Another approach involves the generation of a current thatopposes EOF. Typically, this is accomplished by applying a thin film ofmetal (e.g., iridium tin oxide or copper) to an external surface of thecapillary. Application of current to the film generates a relativelysmall induced current within the capillary to reverse the EOF (see,e.g., Schasfoort, R. B. M., Schlautmann, S., Hendrikse, J., and van denBerg, A., “Field-Effect Flow Control for Microfabricated FluidicNetworks,” Science, 286:942-945 (1999)).

[0085] Placing a porous plug at a location upstream from where sample isintroduced (upstream referring to a direction opposite the flow ofproteins through the capillary) can also be utilized to control EOF. Anexample illustrating the location of the plug is illustrated in FIG. 2Bwhere the capillary 100 extends from the anode reservoir (not shown) atone end and the cathode reservoir at the other end (not shown). Proteinmigration is in the direction of arrow 102 (ie., from the anode tocathode direction). As can be seen, the porous plug 104 is positioned tobe upstream of the trailing edge 106 of the sample once introduced intothe capillary 100. The porous plug 104 is typically formed of apolymeric material and remains relatively stationary duringelectrophoretic runs. Examples of suitable materials from which the plugcan be formed include polymerized acrylamide with diacrylamidecrosslinkers and agarose. Although not intending to be bound by anyparticular theory, the porous plug 104 appears to function as a momentumtransfer barrier by blocking replacement of bulk fluid that in theabsence of the plug 104 would move toward the cathode reservoir.

[0086] In some methods, such as those containing large amounts ofprotein and/or a large number of different proteins, EOF should bereduced to very low levels to allow proteins the opportunity to focusbefore the electrophoretic medium begins eluting from the capillary dueto EOF. In certain methods an EOF of=0.5×10⁻⁶ cm²/V-s (at pH 8.6, and 25mM TRIS-phosphate) has been found to allow ample time for the necessaryfocusing of proteins before sample elutes from the capillary. Methodsdescribed above can reduce EOFs to these levels.

[0087] Thus, the foregoing approaches enable fractions to be collectedaccording to different criteria. Electroelution techniques, for example,can be used to collect fractions having a defined pH range. EOF elutionand pressure elution, in contrast, can be used to separate fractionsaccording to time of elution. Other techniques can also be utilized toelute resolved proteins after CIEF (see, e.g. Kilar, F., “IsoelectricFocusing in Capillaries,” in CRC Handbook or Capillary Electrophoresis:A Practical Approach, CRC Press, Inc., chapter 4, pp. 95-109 (1994)).The controlled elution techniques are useful for enhancingreproducibility, an important factor in comparative and diagnosticmethods. Such techniques also provide improved tolerance of highabundance proteins as compared to methods relying on spatial separation.

[0088] C. Capillarv Zone Electrophoresis (CZE)

[0089] 1. General

[0090] Capillary zone electrophoresis is an electrophoretic methodconducted in free solution without a gel matrix and results in theseparation of molecules such as proteins based upon their intrinsiccharge-to-mass ratio. One advantage to CZE methods is the ability to runwith solvent systems that would normally be incompatible with typicalwater soluble gel matrices. Nonaqueous or water miscible solvent systemscan be used to improve the solubility of hydrophobic and membrane boundproteins that would normally not be resolved by gel electrophoreticmethods. General methods for conducting the method are described, forexample, by McCormick, R. M. “Capillary Zone Electrophoresis ofPeptides,” in CRC Handbook of Capillary Electrophoresis: A PracticalApproach, CRC Press Inc., chapter 12, pp. 287-323 (1994); Jorgenson, J.W. and Lukacs, K. D., J. High Resolut. Chromatogr. Commun., 4:230(1981); and Jorgenson, J. W. and Lukacs, K. D., Anal. Chem. 53:1298(1981)), each of which is incorporated by reference in its entirety.

[0091] 2. System and Solutions

[0092] In general, the capillaries described above for CIEF are alsosuitable for conducting CZE methods. Often the capillaries have internaldiameters of about 50 to 100 microns. Buffer composition and pH cansignificantly influence separations since separations in CZE are basedupon charge-to-mass ratios and the charge of a protein is dependent uponthe pH of the surrounding solution. At the extremes of pH (i.e., below 2and above 10) it is typically difficult to achieve resolution ofproteins because all residues are either fully protonated ordeprotonated and many proteins have a similar number of acidic and basicresidues per unit mass. Selectivity is typically enhanced atintermediate pH. For proteins having a relatively high percentage ofacidic residues, selectivity can often be enhanced near pH 4.5. Forthose proteins having a high concentration of basic residues,selectivity can be enhanced near pH 10.

[0093] In CZE, solutions at the anode and cathode are typically thesame. The buffer utilized can be essentially any buffer, the choice ofbuffer being controlled in part by the pH range at which theelectrophoretic method is conducted and its influence on the detectornoise. Examples of useful buffers at low pH include, but are not limitedto, phosphate and citrate; useful buffers at high pH includeTris/Tricine, borate and CAPS (3-(cyclohexylamino)-1-propane sulfonicacid). Further guidance regarding suitable buffers and buffer additivesis described by McCormick, R. M. “Capillary Zone Electrophoresis ofPeptides,” in CRC Handbook of Capillary Electrophoresis: A PracticalApproach, CRC Press Inc., chapter 12, pp. 287-323 (1994).

[0094] 3. Elution

[0095] Elution can be accomplished utilizing some of the same methodsdescribed above for CIEF, namely pressure and EOF. As with CIEF,controlling EOF can be important in certain methods to preventelectrophoretic medium containing protein from eluting from thecapillary before the proteins within the loaded sample have had anopportunity to separate. EOF can be controlled using the same methodsutilized for controlling EOF in CIEF methods (e.g., coating the internalwalls of the capillary, using a porous plug and generating an inducedfield to counteract EOF). Regulating and carefully selecting the pH andionic strength of the electrophoretic medium is another technique thatcan be used. Because EOF results from ionization of the silanol groupson the interior capillary surface, by conducting CZE at relatively lowpH (e.g., pH 2-5, more typically about pH 3-4) the number of silanolgroups that are ionized is reduced. Such a reduction reduces EOF. Toprevent sample elution prior to complete separation, in certain analysesthe EOF should be reduced to <1×10⁻⁴ cm²/V-s (at pH 8.6 and 25 mMTRIS-phosphate buffer). EOFs of this level can be obtained using themethods just described.

[0096] Another approach that is described more fully below in thedetection and labeling section is to label proteins in the sample priorto injecting the sample containing the protein into the capillary. Byselecting labels that preferentially react with certain functionalgroups such as amino or carboxyl groups, the charge-to-mass ratio ofcertain proteins can be altered. Such alterations can improve theresolution of proteins during electrophoresis as well as improve theirdetectability. (See Examples 1 and 2 below).

[0097] D. Capillary Gel Electrophoresis (CGE)

[0098] 1. General

[0099] Capillary gel electrophoresis refers to separations of proteinsaccomplished by sieving through a gel matrix, resulting in theseparation of proteins by size. In one format, proteins are denaturedwith sodium dodecyl sulfate (SDS) so that the mass-to-charge ratio isdetermined by this anionic surfactant rather than the intrinsicmass-to-charge ratio of the protein [50, 2]. This means that proteinscan be separated solely on the basis of size without charge factoringinto the degree of separation. The application of general SDS PAGEelectrophoresis methods to capillary electrophoresis (CGE) is described,for example, by Hjerten, S., “Free zone electrophoresis,” Chromatogr.Rev., 9:122 (1967).

[0100] 2. System and Solutions The type of capillaries and their sizeare generally as described above for CZE. A variety of different bufferscan be used, including commercially available buffers such as the “eCAPSDS” buffers manufactured by Beckman (see, also, 51, 30, 9 and 5).Various buffer additives can be utilized to increase resolution. Suchadditives, include, but are not limited to, small amounts of organicsolvents, such as N,N-dimethylformamide, cyclohexyldiethylamine,dimethoxytetraethylene glycol and other polyols (e.g., ethylene glycoland polyethylene glycol) (see, e.g., [2] and [3]). The use of suchsolvents can improve the solubility of proteins in aqueous solution andenhance protein stability against thermal denaturation, [52] depress theelectroosmotic flow in CZE and CGE [53], alter the electricaldouble-layer thickness at the capillary wall to inhibit protein bindinginteractions [47] and increase the viscosity of the running buffer whichdepresses the electroosmotic flow. Solvents utilized should becompatible with the polymer matrix inside the capillary.

[0101] Isotachophoresis (IPE) can be used in certain methods to increaseresolution of proteins. For a general discussion of IPE, see, forexample, B. J. Wanders and Everaerts, F. M., “Isotachophoresis inCapillary Electrophoresis,” in CRC Handbook of CapillaryElectrophoresis: A Practical Approach, chap. 5, pp. 111-127 (1994),which is incorporated by reference in its entirety. The velocity of acharged molecule moving through a capillary under a constant fieldstrength depends on its relative mobility, which is a function of themass/charge of the molecule, temperature, and viscosity of the mediumthrough which it is moving. However, in the absence of an adequateconcentration of highly mobile ions upstream of the sample ions, all theions eventually have to migrate at the speed of the slowest ion once theelectric field reaches a steady-state inside the capillary. Thiscondition causes the anions to stack in order of their relativemobilities at the interface of the leading and terminating buffers.

[0102] Under SDS denaturing conditions, all the proteins present in thesample have nearly identical mass/charges. By using a higher mass/chargeanion in the terminal buffer, one can force the proteins to move at aconstant slow speed through the capillary. This has two effects. First,proteins “stack” at the terminal edge of the leading buffer increasingtheir effective concentration inside the capillary. Second, anyseparation between proteins is based on their size. Therefore, the useof a hybrid IPE-CGE method in which the IPE is used for sample“stacking” can improve the resolution possible in the subsequent CGEseparation in some methods.

[0103] Various terminal buffer systems can be utilized in conjunctionwith IPE methods. In one system, ε-aminocaproic acid (EACA) is used asthe terminal electrolyte because it has a high mass/charge at high pH(>6). Tris(hydroxyethyl)aminomethane (TRIS) citrate at 0.05M is used asthe leading buffer at pH=4.8 and as an intermediate stacking buffer atpH=6.5. The sample proteins initially “stack” because EACA has a verylow mobility in the pH 6.5 stacking buffer, but once the protein “stack”and EACA reach the lower pH leading buffer, the mobility of the EACAsurpasses that of the proteins and separation commences (see, e.g.,[57]). This system can be used to create a hybrid single columnIPE-CPAGE system.

[0104] A 2 buffer system for IPE for the separation of proteins involvesdissolving sample in 0.01M acetic acid, which is also used as theterminal electrolyte. The leading and background buffer was 0.02Mtriethylamine-acetic acid solution at pH 4.4. The sample in terminalbuffer is sandwiched between the leading and background buffer. IPEcontinues until the background buffer overtakes the leading edge of theterminal buffer, at which point IPE stops and separation begins (see,e.g., [58]).

[0105] Another IPE approach that can be accomplished with any runningbuffer is to dissolve the sample in the running buffer but diluted to alower ionic strength. This causes an increase in the electricalresistance in the capillary where the sample plug is loaded andcorrespondingly faster movement of the ions present in the sample matrixto running buffer boundary. The optimal ionic strength differencebetween the sample matrix and the running buffer is typically about10-fold (see, e.g., [43]).

[0106] 3. Elution

[0107] In general, the discussion of elution for CZE applies to CGE.Elution can be accomplished utilizing pressure and EOF. As with CIEF andCZE, controlling EOF can be important in certain methods to preventelectrophoretic medium containing protein from eluting before theproteins within the applied sample have had an opportunity to separate.The methods described supra for CIEF and CZE can be used to control EOFat desired levels. To prevent sample elution prior to completeseparation, in certain analyses the EOF should be reduced to <1×10⁻⁴cm²/V-s (at pH 8.6 and 25 mM TRIS-phosphate buffer). EOF can be reducedto this range, for example, by controlling the pH of the buffer, bygeneration of a counteracting induced field, capillary coatings and aporous gel plug.

[0108] E. Labeling and Detection

[0109] As indicated in FIG. 1, electrophoretic solution withdrawn duringthe final electrophoretic separation can be directed toward a detectorfor the detection and quantitation of protein. This arrangement providesconsiderable flexibility with regard to the nature of detection and doesnot limit the methods to the standard gel staining detection techniquescommon in traditional 2-D gel electrophoresis analysis. The detectorneed not be positioned to detect eluted proteins as shown in FIG. 1,however. In other arrangements, the detector is adapted so that it canscan resolved proteins within the separation cavity of the capillarytube itself. To further enhance detection sensitivity, quantitation andreproducibility, proteins are labeled at some point prior to detectionin certain methods. Depending upon the particular label used,signal-to-noise ratios can be achieved which permit the detection of lowabundance proteins.

[0110] Although FIG. 1 depicts a single detector, additional detectorscan be positioned to detect proteins eluting from all or at leastmultiple capillaries utilized in the different electrophoretic methods.One suitable arrangement, for example, involves utilizing a UV/VISdetector to detect eluting proteins from early and/or intermediatemethods, in part to monitor the amount of protein being collected withina fraction. Labeling can then be conducted immediately prior to thefinal step with subsequent detection of labeled protein from the finalcapillary. If labeling is conducted at some earlier stage, then adetector (or detectors), can detect labeled protein after all subsequentelectrophoretic methods in the series.

[0111] Proteins can be detected utilizing a variety of methods. Oneapproach is to detect proteins using a UV/VIS spectrometer to detect thenatural absorbance by proteins at certain wavelengths (e.g., 214 or 280nm). In other approaches, proteins in the various fractions can becovalently labeled through a variety of known methods with chromagenic,fluorophoric, or radioisotopic labels. A wide variety of chemicalconstituents can be used to attach suitable labels to proteins.Chemistries that react with the primary amino groups in proteins(including the N-terminus) include: aryl fluorides [69, 70, 71, 72],sulfonyl chlorides [73], cyanates [74], isothiocyanates [75],immidoesters [76], N-hydroxysuccinimidyl esters [77], chlorocarbonates[78], carbonylazides [78], and aldehydes [79, 80]. Examples of chemicalconstituents that preferentially react with the carboxyl groups ofproteins are benzyl halides [78, 81, 82] and carbodiimide [83],particularly if stabilized using N-hydroxysuccinimide [84]. Both ofthese carboxyl labeling approaches are expected to label carboxylcontaining amino acid residues (e.g., aspartate and glutamate) alongwith that of the C-terminus. In addition, tyrosine residues can beselectively [¹³⁵I]-iodinated to allow radiochemical detection.

[0112] As alluded to supra, labeling can be performed at differentpoints prior to detection. In general, however, the decision when tolabel proteins during the overall analysis depends on the particular CEmethods utilized in the series. For example, if CIEF is utilized in oneof the dimensions, then proteins are typically labeled after CIEF.Labeling can alter the pI of the proteins, thereby changing thelocations at which the proteins focus during CIEF. This is notinherently problematic, but it means that the results cannot be comparedwith typical archival 2-D gel electrophoresis results that include anIEF dimension. Thus, to allow comparison with results obtained usingmore traditional approaches, it can be advantageous to delay labelinguntil after CIEF. Certain labels, however, can be utilized that have aminimal effect on the IEF pattern (see, e.g., U.S. Pat. No. 5,320,727,and Jackson, P. et al., Electrophoresis, 9:330-339 (1998), both of whichare incorporated by reference in their entirety).

[0113] If the decision is made not to label protein prior to conductingCIEF for the foregoing reasons, proteins eluting from the CIEF capillaryor proteins in collected fractions can nonetheless be detected bydetecting UV absorbance or intrinsic fluorescence of the aromatic aminoacids (e.g., tryptophan, phenylalanine and tyrosine). If, however,proteins are labeled prior to CIEF, any effect of pH on the absorptivityof the dye or fluorophor label can be mitigated by spin dialysis andbuffer exchange to a constant pH before measurement, or by resuspendinga constant collected volume into a higher ionic strength buffer of aconstant pH.

[0114] Whereas labeling is generally conducted after the CIEF dimensionto avoid altering isoelectric focusing patterns, there can be advantagesto labeling proteins prior to a CZE dimension. Depending upon thecomposition of the protein-containing sample, labeling of proteins canaffect the charge-to-mass ratio of the labeled proteins. For proteinmixtures wherein the proteins have similar charge-to-mass ratios, theuse of labels that preferentially react with particular residues canalter the charge-to-mass ratios sufficiently such that enhancedresolution is achieved. For example, a group of proteins can initiallyhave a similar charge-to-mass ratio. However, if the proteins within thegroup are labeled with a neutral label that reacts primarily with lysinegroups, proteins having a high number of lysine groups will bear morelabel and have a greater alteration in the charge-to-mass ratio thanproteins having a lower number of lysine residues. This differentialeffect can translate into enhanced fractionation during electrophoresis.(See Examples 1 and 2).

[0115] A variety of labels that preferentially react with specificresidues are available for use. The reactive functionality on the labelis selected to ensure labeling of most or all of the components ofinterest. For example, sulfophenylisothiocyanate can be used toselectively label lysine residues [20], altering their charge frompositive (below a pH of 10) to negative (above a pH of 0.5). Similarly,phenylisothiocyanate can be used to neutralize the lysine and N-terminalpositive charges at all pH. Dansyl chloride can be used to lower the pHat which lysine and N-terminal residues carry a net positive charge. Theaddition of amino functional alkyl ammonium salts to aspartic andglutamic acid residues, such as through carbodiimide coupling, alterstheir charge from negative to positive at low pH.

[0116] There is somewhat greater flexibility in the time at whichproteins are labeled relative to CGE. In some instances, pre-labeling isadvantageous in that the separation can be viewed as it occurs and inthat detection can be performed at the end without further labeling.With pre-labeling there are also fewer fractions of proteins to label.Pre-labeling methods for ID-PAGE are described, for example, by Hames,B. D. in Gel Electrophoresis of Proteins: A Practical Approach, (Hames,B. D. and Rickwood, P., Eds.) 2^(nd) ed., pp. 67-68, Oxford UniversityPress, Oxford (1990) and Rose, D. R. J. and J. W. Jorgensen,“Post-capillary fluorescence detection in capillary zone electrophoresisusing o-phthaldialdehyde,” J. Chromatogr., 447:117 (1988), which areincorporated by reference in their entirety.

[0117] Although certain types of labels are preferred to enhanceseparation in CZE methods, in general the label utilized during labelingin one of the methods of the invention can be quite diverse. In generalthe label should not interfere with fractionation during electrophoresisand should emit a strong signal so that even low abundance proteins canbe detected. The label preferably also permits facile attachment toproteins. Suitable labels include, for example, radiolabels,chromophores, fluorophores, electron dense agents, NMR spin labels, achemical tag suitable for detection in a mass spectrometer, or agentsdetectable by infrared spectroscopy or NMR spectroscopy for example.Radiolabels, particularly for spacially resolved proteins, can bedetected using phosphor imagers and photochemical techniques.

[0118] Certain methods utilize fluorophores since various commercialdetectors for detecting fluorescence from labeled proteins areavailable. A variety of fluorescent molecules can be used as labelsincluding, for example, fluorescein and fluorescein derivatives,rhodamine and rhodamine derivatives, naphythylamine and naphthylaminederivatives, benzamidizoles, ethidiums, propidiums, anthracyclines,mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes,chrysenes, stilbenes, anthracenes, naphthalenes, salicyclic acids,benz-2-oxa-1-diazoles (also called benzofurazans), fluorescamines andbodipy dyes. Specific examples of suitable fluorescent labels are listedin Table 1 below. A variety of appropriate fluorescent dyes arecommerciaily available from Sigma Chemical Co. (St. Louis, Mo.) andMolecular Probes, Inc. (Eugene, Oreg.). TABLE 1 Labels and LabelingMethods Linkage Label Source Formed Amine Labeling2,4,6-trinitrobenzenesulfonic acid Aldrich Aryl amineLissamine ™ rhodamine B sulfonyl Molecular Sulfonamide chloride Probes2′,7′-dichlorofluoroscein-5- Molecular Thiourea isothiocyanate Probes4,4-difluoro-5,7-dimethyl-4-bora- Molecular Amide3a,4a-diaza-s-indacene-3-propionic Probes acid, sulfosuccinimidyl esterNahthalene-2,3-dicarboxylaldehyde Molecular Isoindole Probes CarboxylLabeling 5-(bromomethyl)fluorescein Molecular Ester ProbesN-cyclohexyl-N′-(4-(dimethylamino) Molecular N-Acylureanaphthyl)carbodiimide Probes 1-ethyl-3-(3-dimethylaminopropyl)- PierceAmide carbodiimide hydrochloride with N- Aldrich hydroxysuccinimide and5- Molecular aminofluorescein Probes

[0119] In some instances, the proteins separated by the methods of theinvention are subjected to further analysis by mass spectroscopy. Insuch instances, particular labels can be utilized to enhance separationof mass fragments into certain parts of the mass spectrum. Suitablelabels in such methods are set forth more fully in copending applicationSer. No. ______, entitled “Methods for Protein Sequencing,” havingattorney docket number 020444-000300US, and filed on the same date asthe current application. This application is incorporated herein byreference in its entirety.

[0120] Quantitation of detected signals can be performed according toestablished methods. Peak height and peak area are typically used toquantify the amount of each resolved protein in the finalelectrophoretic dimension. In some methods, the peak height, peak widthat the half height, peak area, and elution time for each peak arerecorded. Peak shape (determined as the height to width ratio) can beused as a measure of the quality of the separation method. Theresolution potential of the method can be determined by correlating theMW of the protein with the elution time (see, e.g., [30] and [11]). Bydividing the overall run time by the average peak width of each proteinan estimate of the total number of proteins that can be resolved by themethod (e.g., proteins separated by at least one peak width can beconsidered a “resolved” protein) can be obtained. The reproducibility ofthe MW estimate can be determined by two methods. In one method, theapparent MW determined for each protein in three replicate runs byestablishing the standard curve from one run and using that curve todetermine the MW based on elution time from each subsequent run arecompared (see, e.g., [21]). In the second approach, the overall error ofthe method is determined from the standard deviation in the slope of thestandard curve created using the data from all three replicate runs.

[0121] The labeling and direct detection approaches that can be usedwith certain methods of the invention can yield improved reproducibilityin the quantification of relative protein expression levels compared tothe staining and imaging methods utilized in conventional 2-D gels.Staining techniques frequently yield poorly quantitative results becausevarying amounts of stain are incorporated into each protein and thestained protein must be detected and resolved against the stainedbackground of the gel or electroblotting substrate. Moreover, since themethods utilize combinations of electrophoretic methods, anelectropherogram that is directly comparable to archived 2-D gel imagedata is still obtained. This means that the methods remain comparable to2-D gel information as compared to other non-electrophoretic basedseparations (e.g., LC/MS/MS).

[0122] F. Exemplary Systems

[0123] The methods of the invention are amenable to a variety ofdifferent electrophoretic methods. The controlled elution techniqueswhereby defined fractions are separated spatially, physically or bytime, and the labeling and detection methods can be utilized in a numberof different electrophoretic techniques. As noted above, the number ofelectrophoretic methods linked in series is at least two, but caninclude multiple additional electrophoretic methods as well. In someinstances, each electrophoretic method in the series is different;whereas, in other instances certain electrophoretic methods are repeatedat different pH or separation matrix conditions.

[0124] Despite the general applicability of the methods, as noted aboveCIEF, CZE and CGE methods are specific examples of the type ofelectrophoretic methods that can be utilized according to the methods ofthe invention. In certain methods, only two methods are performed.Examples of such methods include a method in which CIEF is performedfirst followed by CGE. Labeling is typically performed after CIEF withdetection subsequent to elution of protein from the CGE capillary.Protein eluting from the CIEF capillary can be detected using a UVVISspectrometer at 214 or 280 nm, for example. In another system, the firstmethod is CZE and the final method is CGE. With this arrangement,labeling is typically performed prior to CZE to enhance resolution asdescribed supra. Detection generally is not performed until thecompletion of the final electrophoretic separation. A third usefulapproach involves initially conducting CIEF followed by CZE and CGE.Labeling for such a system is typically done after CIEF and before CZE.Labeling at this point in the overall method avoids alteration of CIEFpatterns (see supra) and allows for greater resolution during CZE.Detection is generally conducted at the conclusion of CGE (i.e., withresolved protein within the capillary or after the proteins have elutedfrom the capillary). These are specific examples of systems that can beutilized; it should be understood that the invention is not limited tothese particular systems. Other configurations and systems can bedeveloped using the techniques and approaches described herein.

[0125] IV. Samples

[0126] The methods of the invention can be used with a wide range ofsample types. Essentially any protein-containing sample can be utilizedwith the methods described herein. The samples can contain a relativelysmall number of proteins or can contain a large number of proteins, suchas all the proteins expressed within a cell or tissue sample, forexample.

[0127] Samples can be obtained from any organism or can be mixtures ofsynthetically prepared proteins or combinations thereof. Thus, suitablesamples can be obtained, for example, from microorganisms (e.g.,viruses, bacteria and fungi), animals (e.g., cows, pigs, horses, sheep,dogs and cats), hominoids (e.g., humans, chimpanzees, and monkeys) andplants. The term “subject” as used to define the source of a sampleincludes all of the foregoing sources, for example. The term “patient”refers to both human and veterinary subjects. The samples can come fromtissues or tissue homogenates or fluids of an organism and cells or cellcultures. Thus, for example, samples can be obtained from whole blood,serum, semen, saliva, tears, urine, fecal material, sweat, buccal, skin,spinal fluid, tissue biopsy or necropsy and hair. Samples can also bederived from ex vivo cell cultures, including the growth medium,recombinant cells and cell components. In comparative studies toidentify potential drug or drug targets (see infra), one sample can beobtained from diseased cells and another sample from non-diseased cells,for example.

[0128] Sample preparation for the different electrophoretic techniquesis set forth above. If the sample contains cellular debris or othernon-protein material that might interfere with separation duringelectrophoresis, such materials can be removed using any of a variety ofknown separation techniques including, for example, forcibly exuding thesample through sieve material, filtration and centrifugation. Sampleswhose ionic strength is particularly high can be desalted usingestablished techniques such as dialysis and dilution andreconcentration.

[0129] In some instances in which the sample contains salts or otherinterfering components, buffer exchange can be performed to improve IPE“stacking” and improve reproducibility in elution times and peak shapesfor electrophoretic methods. One useful way to implement dialysis toremove interfering compounds is to collect fractions directly in thedialysis chamber of a spin dialysis tube (Gilson/Amicon). The sample canthen be spin dialyzed and resuspended in a 10-fold dilution of therunning buffer to be utilized in the next electrophoretic separation ofthe series. This procedure has the advantages that:

[0130] (1) in the case of CIEF, larger volumes of buffers can be usedduring electroelution of each fraction without diluting the proteins ineach fraction, (2) the same sample volume can be used for each fractioninjected into the second dimension and (3) smaller more concentratedsample volumes can be used in the second dimension because the dialyzedproteins can be resuspended in almost any buffer volume after dialysis.

[0131] V. Variations

[0132] A. Further Analysis

[0133] The methods of the invention need not end with the lastelectrophoretic method of the series. As illustrated in FIG. 1, resolvedproteins can be further analyzed by non-electrophoretic methods.Examples of such methods include infra-red spectroscopy, nuclearmagnetic resonance spectroscopy, UV/VIS spectroscopy and complete orpartial sequencing. Coupling the current electrophoretic-based method tovarious mass spectroscopy (MS) methods is one specific example offurther analysis that can be conducted. A variety of mass spectraltechniques can be utilized including several MS/MS methods andElectrospray-Time of Flight MS methods (see, e.g., [61], [62], [63], and[64]). Such methods can be used to determine at least a partial sequencefor proteins resolved by the electrophoretic methods such as a proteinsequence tag (for a discussion or protein sequence tags, see, e.g., [65]and [66]). Further discussion regarding combining the electrophoreticseparations described herein with mass spectral analysis is set forth inU.S. provisional application 60/130,238 entitled “Rapid and QuantitativeProtein Expression and Sequence Determination,” filed Apr. 20, 1999, andto which this application claims benefit and which is incorporated byreference in its entirety. Other mass spectral methods that can becombined with the methods of the present invention are described incopending U.S. application Ser. No. ______, entitled “Methods forProtein Sequencing,” and having attorney docket number 020444-000300US,and copending U.S. application Ser. No. ______, entitled “PolypeptideFingerprinting Methods and Bioinformatics Database System, and havingattorney docket number 020444-000100US, both filed on the same date asthe current application and both being incorporated by reference intheir entirety.

[0134] B. Microfluidic Systems

[0135] 1. Examples of Configurations

[0136] In another variation, the capillaries are part of or formedwithin a substrate to form a part of a microfluidic device that can beused to conduct the analyses of the invention on a very small scale andwith the need for only minimal quantities of sample. In these methods,physical fractions of samples typically are not collected. Instead,resolved proteins are separated spatially or by time. Methods forfabricating and moving samples within microfluidic channels orcapillaries and a variety of different designs have been discussedincluding, for example, U.S. Pat. Nos. 5,858,188; 5,935,401; 6,007,690;5,876,675; 6,001,231; and 5,976,336, all of which are incorporated byreference in their entirety.

[0137] An example of a general system 150 that can be used with themethods of the present invention is depicted in FIG. 3A. The capillariesor channels are typically formed or etched into a planar support orsubstrate. A separation capillary 152 extends from an anode reservoir154 containing anolyte to a cathode reservoir 156. The anode reservoir154 and the cathode reservoir 156 are in electrical contact with ananode and cathode 158, 160, respectively. A sample injection channel 162runs generally perpendicular to the separation capillary 152 and one endintersects at an injection site 164 slightly downstream of the anodereservoir 154. The other end of the sample injection capillary 162terminates at a sample reservoir 166, which is in electricalcommunication with a sample reservoir electrode 168. A detector 170 ispositioned to be in fluid communication with electrophoretic mediumpassing through the separation capillary 152 and is positioneddownstream of the sample injection site 164 and typically somewhatupstream of the cathode reservoir 156. In this particular configuration,fractions are withdrawn into the cathode reservoir 156. Movement ofelectrophoretic medium through the various channels is controlled byselectively applying a field via one or more of the electrodes 158, 160168. Application of a field to the electrodes controls the magnitude ofthe EOF within the various capillaries and hence flow through them.

[0138] An example of another configuration is illustrated in FIG. 3B.This system 180 includes the elements described in the system shown inFIG. 3A. However, in this arrangement, spacially or temporally resolvedfractions can be withdrawn at multiple different locations along theseparation capillary 152 via exit capillaries 172 a, 172 b and 172 c.Each of these capillaries includes a buffer reservoir 176 a, 176 b, 176c, respectively, and is in electrical communication with electrodes 174a, 174 b, 174 c, respectively. Movement of electrophoretic medium alongseparation capillary 152 and withdrawal of fractions therefrom into theexit capillaries 172 a, 172 b and 172 c can be controlled by controllingwhich electrodes along the separation capillary 152 and which of theexit capillary electrodes are activated. Alternatively, or in addition,various microfluidic valves can be positioned at the exit capillaries172 a, 172 b and 172 c to control flow. Typically, additional detectorsare positioned at the various exit capillaries 172 a, 172 b and 172 c todetect protein in fractions withdrawn into these capillaries.

[0139] The configuration illustrated in FIG. 3B can be used in a numberof different applications. One example of an application for which thistype of system is appropriate is a situation in which the type ofsamples being examined have been well characterized. If for example,certain fractions of proteins of interest have been previouslyestablished to fractionate at a particular location in the separationcapillary 152, then the exit capillaries 172 a, 172 b and 172 c can bepositioned at those locations to allow for selective removal of theprotein fraction(s) of interest.

[0140] In still another configuration, multiple exit capillaries branchfrom the end of the separation capillary 152 near the cathode reservoir156, each exit capillary for withdrawing and transporting separatefractions. In this configuration also, withdrawal of fractionatedprotein from the separation capillary can be controlled by regulatingEOF within the various capillaries and/or by microfluidic valves.

[0141] Other components necessary for conducting an electrophoreticanalysis can be etched into the support, including for example thereservoirs, detectors and valves discussed supra.

[0142] 2. Substrates

[0143] The substrate upon which the capillary or micro-channel networkof the analytical devices of the present invention are formed can befabricated from a wide variety of materials, including silicon, glass,fused silica, crystalline quartz, fused quartz and various plastics, andthe like. Other components of the device (e.g., detectors andmicrofluidic valves) can be fabricated from the same or differentmaterials, depending on the particular use of the device, economicconcerns, solvent compatibility, optical clarity, mechanical strengthand other structural concerns. Generally, the substrate is manufacturedof a non-conductive material to allow relatively high electric fields tobe applied to electrokinetically transport the samples through thevarious channels.

[0144] In the case of polymeric substrates such as plastics, thesubstrate materials can be rigid, semi-rigid, or non-rigid, opaque,semi-opaque or transparent, depending upon the use for which thematerial is intended. Plastics which have low surface charge whensubjected to the electric fields of the present invention and thus whichare of particular utility include, for example, polymethylmethacrylate,polycarbonate, polyethylene terepthalate, polystyrene or styrenecopolymers, polydimethylsiloxanes, polyurethane, polyvinylchloride,polysulfone, and the like.

[0145] Devices which include an optical or visual detector are generallyfabricated, at least in part, from transparent materials to facilitatedetection of components within the separation channel by the detector.

[0146] 2. Channel Structure/Formation

[0147] The size and shape of the channels or capillaries formed in thesubstrate of the present devices can have essentially any shape,including, but not limited to, semi-circular, cylindrical, rectangularand trapezoidal. The depth of the channels can vary, but tends to beapproximately 10 to 100 microns, and most typically is about 50 microns.The channels tend to be 20 to 200 microns wide.

[0148] Manufacturing of the channels and other elements formed in thesurface of the substrate can be carried out by any number ofmicrofabricating techniques that are known in the art. For example,lithographic techniques may be employed in fabricating glass or quartzsubstrates, for example, using established methods in the semiconductormanufacturing industries. Photolithographic masking, plasma or wetetching and other semiconductor processing technologies can be utilizedto create microscale elements in and on substrate surfaces.Alternatively, micromachining methods, such as laser drilling,micromilling and the like, can be utilized. Manufacturing techniques forpreparing channels and other elements in plastic have also beenestablished. These techniques include injection molding techniques,stamp molding methods, using for example, rolling stamps to producelarge sheets of microscale substrates, or polymer microcastingtechniques, wherein the substrate is polymerized within a micromachinedmold.

[0149] Further guidance regarding other designs and methods for usingsuch microfluidic devices such as described above can be found, forexample, in U.S. Pat. Nos. 5,858,188; 5,935,401; 6,007,690; 5,876,675;6,001,231; and 5,976,336, all of which are incorporated by reference intheir entirety.

[0150] C. Preliminary Separation by Non-Electrophoretic Technique

[0151] The methods can also include an initial separation by anon-electrophoretic technique prior to commencing the electrophoreticseparations. Essentially any type of technique capable of separatingproteins can be utilized. Suitable methods include, but are not limitedto, fractionation in a sulfate gradient, HPLC, ion exchangechromatography and affinity chromatography.

[0152] VI. Exemplary Utilities

[0153] The methods and apparatus of the invention can be utilized todetect, characterize and/or identify many proteins (e.g., hundreds orthousands of proteins in some methods) by controlling elution offractionated proteins and utilizing various labeling and detectiontechniques. Consequently, the methods have multiple utilities including,but not limited to, various analytical applications (e.g., monitoringcertain protein levels as a function of external stimuli, or detectingspecific proteins in complex compositions for identification purposes),clinical applications (e.g., detecting and/or monitoring compositions ofnormal and diseased cells and tissues, diagnosing or monitoring disease,testing drug candidates for therapeutic efficacy and toxicity testing)and molecular biology and genetic research (e.g., characterizing ormonitoring molecular expression levels of gene products and determiningthe effects of the addition, mutation, deletion or truncation of aparticular gene). In general, the methods and apparatus have utility inproteome research.

[0154] More specifically, the invention can be used in the developmentof protein databases in which, for example, proteins expressed underparticular conditions are isolated, quantified, and identified. Usingthe controlled elution and detection methods described herein, certainmethods can be utilized to determine and catalog a variety of chemicaland physical characteristics of the resolved proteins, including, butnot limited to, pI, and/or apparent molecular weight and/or relativeabundance of proteins within a sample. This information can be furthercross referenced with a variety of information regarding the source ofthe sample and the method by which it was collected. Examples of suchinformation include genus, species, age, race, sex, environmentalexposure conditions, subject's health, tissue type, method of samplecollection and method of sample preparation prior to electrophoresis.

[0155] The methods also have value in a variety of comparative studiesthat can be utilized to identify potential drug targets and/orcandidates. For example, the methods can be utilized to identifyproteins that are differentially expressed in diseased cells as comparedto normal cells. Such differentially expressed proteins can serve astargets for drugs or serve as a potential therapeutic. In a relatedfashion, the methods can be used in toxicology studies to identifyproteins that are differentially expressed in response to particulartoxicants. Such differentially expressed proteins can serve as potentialtargets or as potential antidotes for particular toxic compounds orchallenges. The detection and labeling techniques of the invention canfacilitate such investigations because these techniques enable even lowabundance proteins to be detected and because enhanced reproducibilitymakes it easier to identify real differences in expression betweendifferent samples.

[0156] Proteornic studies using certain methods of the invention candetect mutations that result in premature termination of the genetranscript or in amino acid substitutions in the resulting gene product.The methods can also detect post translational modification eventsassociated with disease that are not readily detectable or possible todetect using functional genomics. For example, proteomic methods candetect differences in protein folding, glycosylation patterns,phosphorylation events, and degradation rates.

[0157] The results of comparative studies are transferable to a varietyof diagnostic applications. For example, the “marker” or “fingerprint”proteins identified during comparative studies as being characteristicof a particular disease can be used to diagnosis individuals todetermine if they have the disease correlated with the marker. Thesemarkers can also be used in medical screening tests. Once such protemshave been identified, it is not necessary to examine all fractions.Instead, only those fractions potentially containing the marker proteinsneed be examined. The reproducibility of the methods facilitates suchanalyses. For systems integrated onto a chip or support (see supra),capillaries can be positioned at the appropriate locations along theseparation cavity to withdraw only the relevant fractions potentiallycontaining the marker protein(s) of interest.

[0158] As an example of a diagnostic application, proteomic analysis canbe utilized in identifying diagnostic markers (e.g., cell surfaceantigens or serum proteins) for immunodiagnostic assays. Purifiedsamples of putative diagnostic proteins are recovered during proteomicanalysis, and can be used to generate antibodies having specific bindingaffinity to the proteins. Such antibodies can be used to understand thelink between the marker protein and the disease through immunologicalstaining to localize the protein in diseased cells or to rapidly screenpatients for the presence of the protein, showing its statistical linkto the disease.

[0159] The methods of the invention have further utility in conductingstructure activity studies. For instance, the methods can be used todetermine the effect that certain chemical agents or combination ofagents have on protein expression patterns. Alterations to the agent orcombination can then be made and protein expression reassessed todetermine what effect if any the alteration has on protein expression.Such studies can be useful, for example, in making derivatives of a leadcompound identified during initial drug screening trials.

[0160] Metabolic engineering studies can also be conducted using themethods of the invention. In such studies, a gene can be geneticallyengineered to include certain changes or the promoter of a gene begenetically engineered to increase or decrease its relative expressionlevel. The methods described herein can then be used to determine whateffect, if any, the genetically engineered changes have on proteinsother than the protein encoded by the genetically engineered gene.

[0161] The following examples are offered to illustrate, but no to limitthe claimed invention.

EXAMPLE 1 CZE Separation of Unlabeled Proteins

[0162] Each of five proteins (see Table 2) were obtained fromSigma-Aldrich and were suspended at 5 mg/ml in an aqueous denaturingsample buffer consisting of 25 mM tris(hydroxymethyl)aminomethanephosphate (pH 4.0), 0.5% by weight IGEPAL CA-630 (obtained fromSigma-Aldrich, Cat # 13021), and 1% by weighttris(2-carboxyethylphosphine)hydrochloride (TCEP, obtained from Pierce,Cat # 20490ZZ). The protein samples were denatured in this sample bufferby heating at 95° C. for 15 min. Each of the five denatured proteinsamples were diluted into a cZE sample buffer to create a final solutionconsisting of 25 mM tris(hydroxymethyl)aminomethane phosphate buffer (pH4.0), 8 M Urea, and a final concentration of 0.2 mg/ml of each of thefive proteins. Control samples were also prepared of each denaturedprotein separately at 0.5 mg/ml final concentration in the same samplebuffer. TABLE 2 Protein Standards Protein Cat # pl MW (kDa) Hen eggwhite conalbumin C 0755 6.0, 6.3, 6.6 76.0 Bovine serum albumin B 42875.4, 5.5, 5.6 66.2 Carbonic anhydrase II T 6522 4.5 21.5 Rabbit muscleGAPDH G 2267 8.3, 8.5 36.0 Bovine ribonuclease A R 5503 9.6 13.7

[0163] The mixed protein sample and each of the control samples were runby CZE in a 60 cm×75 μm fused silica capillary (Beckman Coulter). An 800μm detection window was located 50 cm from the anodic end of thecapillary. A 160 nl sample volume was pressur injected at the anodic endand the separations conducted at 500 V/cm in a 25 mM TRIS-phosphate and8 M urea running buffer at pH 4.0. Protein detection was accomplished byUV adsorption at 214 nm.

[0164] The individual unlabeled proteins were not resolved under theseconditions (see FIG. 4). The electrophoretic mobility of each proteinwas determined from replicate runs of the individual protein controls(FIG. 5) and correlated with the predicted mass to charge ratio of theproteins at pH 4.0 (FIG. 6). The mass to charge ratio for each of theunlabeled proteins was determined from the published protein sequencesobtained through Genbank in the manner described by Canter, C. R. andSchimmel, P. R., Biophysical Chemistry, W.H. Freeman and Co., New York,(1980), which is incorporated by reference in its entirety.

EXAMPLE 2 CZE Separation of Labeled Proteins, with Fraction Collection

[0165] Each of the five proteins described in Example 1 was suspended at10 mg/ml in a denaturing buffer containing 1% by weight of sodiumdodecyl sulfate and 1% by volume 2-mercaptoethanol. The proteins weredenatured in this buffer by heating at 95° C. for 15 min. The denaturedprotein samples were labeled with 4-sulfophenylisothiocyanate (SPITC)obtained from Sigma-Aldrich (Cat # 85,782-3) and used as supplied.Labeling was accomplished by adding 0.01 ml of triethylamine, 0.01 ml of2 M acetic acid and 0.02 ml of a 10% by weight solution of SPITC inwater to 0.1 ml of each denatured protein sample. The reaction mixturewas heated at 50° C. for 24 h.

[0166] A quantity of 0.05 ml of each of the SPITC-labeled proteinstandards was mixed together and separated by cZE as described inExample 1, with the exception that the pH of the separation buffer wasadjusted to 3.0. The individual SPITC-labeled proteins were resolved(FIG. 7). Thus, this example taken in view of the results for Example 1in which unlabeled proteins were poorly resolved demonstrates thepositive effect that labeling can have when done prior to a cZEseparation. Fractions were collected by electroelution into separatevials containing the separation buffer at the times indicated. Theidentities of the SPITC-labeled proteins were determined by subsequentcGE analysis of the fractions.

EXAMPLE 3 CIEF First Dimension Separation with Fraction Collection

[0167] Bovine Serum Albumin, Carbonic Anhydrase, and Conalbumin wereused as supplied from Sigma-Aldrich (Table 2). Each protein wasdenatured as described in Example 1. A 0.01 ml aliquot of each denaturedprotein sample was added to 0.2 ml of the CIEF focusing buffer. The CIEFfocusing buffer consisted of 0.4% by weight hydroxymethyl cellulosesolution (Beckman-Coulter eCAP CIEF Gel Buffer, Cat # 477497) containing1% by volume pH 3-10 Ampholytes (Fluka, Cat # 10043) and 1% by weight3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate.

[0168] A poly(ethylene glycol)-coated 60 cm long 0.1 mm internaldiameter fused silica capillary (Supelcowax 10, Supelco, Cat # 25025-U)was filled with the protein sample in the focusing buffer. The capillarycontents were focused between 10 mM phosphoric acid and 20 mM NaOHreservoirs for 7.5 min at 500 V/cm and 25 C. A 0.5 psi pressure gradientwas then applied between the anolyte and catholyte reservoirs tofacilitate the elution of the focused proteins in the direction of theelectroosmotic flow.

[0169] The protein peaks were detected by monitoring the ultravioletabsorption at 214 nm through an optical window in the capillarypositioned 50 cm from the low pH end. The current through the capillarywas also monitored (FIG. 8). Fractions (B-G) were collected into 0.05 mlof 20 mM NaOH contained in separate reservoir vials for the timesdepicted (FIG. 8). Only fractions F and G were found to contain protein(see Example 4). Fraction G was found to contain carbonic anhydrase andno conalbumin or bovine serum albumin. Conalbumin and bovine serumalbumin were found to coelute in the peak observed in fraction F. Thisexperiment illustrates the partial separation of a mixture of proteinsin a single dimension. Further resolution was achieved in the seconddimension (see Example 4).

EXAMPLE 4 CGE Second Dimension Separation of CIEF Fractions

[0170] Each of the CIEF fractions (B-G) collected during the CIEFseparation described in Example 3 were evaporated in a Savant ModelSC210A Spin-Vap to a final volume of 0.005 ml to concentrate any proteinpresent in the fraction. A quantity 0.01 ml of SDS sample buffer wasadded to each protein concentrate. The SDS sample buffer consisted of0.1 ml of eCAP SDS sample buffer (Beckman Coulter, Cat # 241525), 0.01ml of eCAP Orange G Reference Marker (Beckman Coulter, Cat # 241524),and 0.09 ml of anhydrous glycerol.

[0171] Each sample was then run in CGE mode using a linearpoly(acrylamide)-coated fused silica capillary 60 cm long with a 100 μminternal diameter. The eCAP SDS 14-200 Gel buffer (Beckman-Coulter Cat#477416) was used for the separation and in both reservoirs. Theseparation was conducted at 20° C. and 500 V/cm for 50 min. Ultravioletdetection of the proteins was accomplished at 214 nm through an opticalwindow positioned 50 cm from the sample injection end of the capillary.Molecular weight calibration was conducted in a separate run using eCAPMW Standards (Beckman-Coulter Cat # 477418) as described by themanufacturer. A 100 sec sample injection at 0.5 psi was used to loadeach sample into the capillary.

[0172] The resulting electropherograms showed no detectable protein inany cIEF fraction except fractions F (FIG. 9) and G (FIG. 10). Themolecular weight of the two proteins seen in fraction F (FIG. 9)correspond to that of bovine serum albumin and conalbumin (Table 2). Themolecular weight of the protein seen in fraction G (FIG. 10)corresponded to that of carbonic anhydrase (Table 1). It is observedthat the second cGE dimension was necessary to fully resolve bovineserum albumin from conalbumin, which were not resolved by a single cIEFmode (Example 3).

EXAMPLE 5 Use of Methods in Proteomics Analysis for DistinguishingBetween Healthy and Cancerous Tissue

[0173] This example illustrates the use of the present invention fordistinguishing between healthy and cancerous tissue. The presentinvention can be used to directly analyze the protein expression patternof healthy and cancerous and metastasized tissues to elucidate patternsof gene expression and translate such relations to the various aspectsof onset, staging and metastases in cancers, such as prostrate, breast,colon and skin.

[0174] The methods of the invention can significantly decrease the timenecessary to conduct functional genomics analysis of the mechanism ofdisease and can lead to the identification of new therapeutic targets,diagnostic markers, and drug products (i.e., where a specific cellularprotein may itself act as a therapeutic agent). By using proteomicanalysis the number of genes that must be investigated is reduced10-fold (from the 50,000 to 150,000 human genes to the 2,000-10,000genes actually being expressed to form proteins in the target tissue).Through quantitative comparison of the protein expression pattern ofhealthy and diseased tissue, the number of candidate genes that may playroles in the progression of the disease is further reduced about100-fold. Finally, through the subsequent generation of protein sequencetags (PTSs; i.e., a partial amino acid sequence) each of the proteinsthat show differential expression can be uniquely identified in a mannerthat allows them to be tracked back to the genome for completesequencing (e.g., mutation detection).

[0175] Initially, tissue samples are obtained from diseased subjects andcontrol subjects (e.g., individuals not known to have the particularcancer being studied). The tissue samples from each individual arehomogenized according to known methods. Depending upon the sample, theresulting homogenate is filtered or centrifuged to remove cellulardebris. Samples are taken from the homogenate and the proteins thereindenatured by adjusting the samples to contain urea (6-8 M), detergent(e.g., 1% by weight sodium dodecyl sulfate) and 1% by weightdithiothreitol. Samples are heated at 95° C. for 15 minutes to speeddenaturation.

[0176] Samples (5 μl) are then electrophoresed by CIEF on a column (75micron inside diameter by 60 cm long). Anolyte is initially 10 mMphosphoric acid and the catholyte is initially 20 mM sodium hydroxide.Separations are conducted at 500 V/cm. Fractions of resolved proteinsare eluted by increasing the sodium chloride concentration of thecatholyte solution from 10 mM to 100 mM in 96 incremental units.Fractions are collected by sequentially inserting the high pH end of thecapillary into 200_l of each salt concentration in catholyte solutioncontained in the wells of a 96 well plate. The separation current isallowed to reequilibrate before the capillary end is moved to the nextfraction.

[0177] Prior to labeling, fractions are concentrated using a rotaryevaporator. Protein in the collected fractions is labeled by reactingthe proteins with fluoroscein isothiocyanate as described in Example 2for sulfophenylisothiocyanate.

[0178] Fractions containing the labeled proteins are separatelyelectrophoresed by CZE. The labeled proteins are diluted into a CZEsample buffer to form a final solution consisting of 25 mMtris(hydroxymethyl)aminomethane phosphate buffer (pH 4.0), 8 M urea, anda final concentration of about 1 mg/ml of protein. The mixed proteinsample and each of the control samples are run in CZE mode in a 60 cm×75μm fused silica capillary (Beckman Coulter). An 800 μm window is located50 cm from the anodic end of the capillary. A 160 nl sample volume ispressure injected at the anodic end and the separations conducted at 500V/cm in a 25 mM TRIS-phosphate and 8 M urea running buffer at pH 4.0.Proteins are eluted by the residual EOF in the capillary. Fractions areagain collected on the basis of elution time in the wells of a 96 wellmicrotiter plate as the capillary is progressively advanced from onewell to the next. Each well contains 200 μl of the cZE separationbuffer. This process is repeated with samples from the other fractionscollected during CIEF.

[0179] Samples from CZE fractions are further resolved by CGE. Fractionsfrom CZE are separately concentrated by rotary evaporation to a finalliquid volume of about 5 μl. The protein sample is isolated fromcrystallized urea by refrigerated (4° C.) centrifugation. Tenmicroliters of SDS sample buffer is added to each vial of proteinconcentrate. The SDS sample buffer consists of 100 μl of eCAP SDS samplebuffer (Beckman Coulter, Cat # 241525), 10 μl of eCAP Orange G ReferenceMarker (Beckman Coulter, Cat # 241524), and 90 μl of anhydrous glycerol.

[0180] Each sample is run in cGE mode using a linearpoly(acrylamide)-coated fused silica capillary 60 cm long with a 100 μminternal diameter. Commercially available eCAP SDS 14-200 Gel buffer(Beckman-Coulter Cat # 477416) is used for the separation and includedin both reservoirs. The separation is conducted at 20° C. and 500 V/cmfor 50 min. Molecular weight calibration is conducted in a separate runusing eCAP MW Standards (Beckman-Coulter Cat # 477418) as described bythe manufacturer. A 100 sec sample injection at 0.5 psi is used to loadeach sample into the capillary. Resolved proteins are detected byfluoroscein fluorescence with a 466 nm laser induced fluorescencedetector.

[0181] The foregoing process is repeated with multiple samples fromdiseased and control subjects, as well as replicate runs with samplesfrom the same subjects. The results are then examined to identifyproteins whose relative abundance varies between diseased and controlsubjects. Such proteins are potential markers for the particular diseaseand/or a drug target or potential drug.

[0182] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent or patent application werespecifically and individually indicated to be so incorporated byreference.

REFERENCES

[0183] 1. Kilár, F., “Isoelectric focusing in capillaries,” in: CRCHandbook of Capillary Electrophoresis: A Practical Approach, Chp. 4,pgs. 95-109 (CRC Press, Boca Raton, Fla., 1994).

[0184] 2. Palmieri, R. and J. A. Nolan, “Protein capillaryelectrophoresis: Theoretical and experimental considerations for methodsdevelopment,” in: CRC Handbook of Capillary Electrophoresis: A PracticalApproach, Chp. 13, pgs. 325-368 (CRC Press, Boca Raton, 1994).

[0185] 3. Wanders, B. J. and F. M. Everaerts, “Isotachophoresis incapillary electrophoresis,” in: CRC Handbook of CapillaryElectrophoresis: A Practical Approach, Chp. 5, pgs. 111-127(CRC Press,Boca Raton, Fla., 1994).

[0186] 4. Anderson, L. and J. Seilhamer, “A Comparison of Selected mRNAand Protein Abundances in Human Liver,” Electrophoresis, 18:533 (1997).

[0187] 5. Hochstrasser, D. F., et al., Anal Biochem., 173:424 (1988).

[0188] 6. O'Farrell, P. H., J. Biol. Chem., 250:4007 (1975).

[0189] 7. Anderson, N. G. and N. L. Anderson, “Twenty years oftwo-dimensional electrophoresis: Past, present and future,”Electrophoresis, 17:443 (1996).

[0190] 8. Lopez, M. F., “2D Electrophoresis of Target Protein Groups andthe Initiation of a Neurological Disease Database,” paper presented atthe IBC Proteomics conference, Coronado, Calif. (Jun. 11-12, 1998).

[0191] 9. Gottlieb, M. and M. Chavko, Anal. Biochem., 165:33 (1987).

[0192] 10. Bio-Rad, “Detection of Proteins in SDS-PAGE: A comparison ofgel staining methods,” EG Bulletin 1820, Rev B (Bio-Rad Laboratories,Hercules, Calif., 1996).

[0193] 11. Schneider, L., “Metabolic uncoupling in Escherichia coliduring phosphate limited growth,” PhD Thesis, Department of ChemicalEngineering, (Princeton University, Princeton, N.J., 1997).

[0194] 12. Merril, C. R., Methods in Enzymology, 182:477 (1990).

[0195] 13. Wilson, C. M., Methods in Enzymology, 91:236 (1983).

[0196] 14. Lee, C., A. Levin and D. Branton, Anal. Biochem., 166:308(1987).

[0197] 15. Dzandu, J. K., J. F. Johnson and G. E. Wise, Anal. Biochem.,174:157 (1988).

[0198] 16. Steinberg, Jones, Haugland and Singer, Anal. Biochem.,239:223 (1996).

[0199] 17. Merril, C. R., N. Arold, D. Taube and W. Ehrhardt,Electrophoresis, 9:255 (1981).

[0200] 18. Garfin, D. E., Methods in Enzymology, 182:425 (1990).

[0201] 19. Laemmli, U. K., Nature, 227:680 (1970).

[0202] 20. Corthals, G. L., M. P. Molloy, B. R. Herbert, K. L. Williams,and A. A. Gooley, “Prefractionation of protein samples prior totwo-dimensional electrophoresis,” Electrophoresis, 18:317 (1997).

[0203] 21. Lopez, M. F., and W. F. Patton, “Reproducibility ofpolypeptide spot positions in two-dimensional electrophoresis ofribosomal and nuclear proteins,” Electrophoresis, 18:338 (1997).

[0204] 22. McKee, A., “The Yeast Proteome,” paper presented at the IBCProteomics conference, Coronado, Calif. (Jun. 11-12, 1998).

[0205] 23. Anderson, L., “Pharmaceutical Proteomics: Targets, mechanismsand function,” paper presented at the IBC Proteomics conference,Coronado, Calif. (Jun. 11-12, 1998).

[0206] 24. Parekh, R. B., “Use of Proteomics in pre-clinicalpharmaceutical research,” paper presented at the IBC Proteomicsconference, Coronado, Calif. (Jun. 11-12, 1998).

[0207] 25. BiORad Molecular Inager FX and PDQuest 2-D analysis softwareseminar, presented at the IBC Proteomics conference, Coronado, Calif.(Jun. 11-12, 1998).

[0208] 26. Patton, W. F., “Defining protein targets for drug discoveryusing Proteomics,” paper presented at the IBC Proteomics conference,Coronado, Calif. (Jun. 11-12, 1998).

[0209] 27. Rarnsby, M., G. Makowski, and E. Khairallah, “Differentialdetergent fractionation of isolated hepatocytes: Biochemical,immunochemical, and two-dimensional gel electrophoresis characterizationof cytoskeletal and noncytoskeletal compartments,” Electrophoresis,15:265 (1994).

[0210] 28. Blomber, A., L. Biomberg, J. Norbeck, S. J. Fey, P.Mose-Larsen, M. Larsen, P. Roepstorff, H. Degand, M. Boutry, A. Poschand A. Görg, Electrophoresis, 16:1935 (1995).

[0211] 29. Corbett, J. M., M. J. Dunn, A. Posch and A. Görg,Electrophoresis, 15:1205 (1994).

[0212] 30. Beckman Instruments, “eCAP SDS 200: Fast, reproducible,quantitative protein analysis,” BR251 B (Beckman Instruments, Fullerton,Calif., 1993).

[0213] 31. Anderson, N. L. et al., “An updated two-dimensional geldatabase of rat liver proteins useful in gene regulation and drugeffects studies, Electrophoresis, 16:1997 (1995).

[0214] 32. Franzen, F., S. Linder, A. A. Alaiya, E. Eriksson, K.Fujioka, A.-C. Bergman, H. Jomvall, G. Auer, “Analysis of polypeptideexpression in benign and malignant human breast lesions,”Electrophoresis, 18:582 (1997).

[0215] 33. Guttman, A., J. A. Nolan and N. Cooke, “Capillary sodiumdodecyl sulfate gel electrophoresis of proteins, J. Chromatogr., 632:171(1993).

[0216] 34. Clauser, K. R., “Managing high-throughput data acquisitionand analysis in LC/MS/MS-based Proteomics,” paper presented at the IBCProteomics conference, Coronado, Calif. (Jun. 11-12, 1998).

[0217] 35. P/ACE™ Laser-induced fluorescence Detectors, BR-8118A(Beckman Instruments, Fullerton, Calif., 1995).

[0218] 36. Wilm, M. and Mann, M., “Analytical properties of thenanoelectrospray ion source,” Anal. Chem., 68:1-8 (1996).

[0219] 37. Steiner, S., “Proteome methods to profile mechanisms oftoxicity,” paper presented at the IBC Proteomics conference, Coronado,Calif. (Jun. 11-12, 1998).

[0220] 38. Arnott, D., “Protein differential display and massspectrometry in the study of congestive heart failure,” paper presentedat the IBC Proteomics conference, Coronado, Calif. (Jun. 11-12, 1998).

[0221] 39. Witzmann, F. A., C. D. Flutz, and J. F. Wyman,“Two-dimensional electrophoresis of precision-cut testis slices:Toxicologic application,” Electrophoresis, 18:642 (1997).

[0222] 40. Hjerten, S., J.-L. Liao and K. Yao, “Theoretical andexperimental study of high-performance electrophoretic mobilization ofisoelectrically focused protein zones, J. Chromatogr., 387:127 (1987).

[0223] 41. Kim, K. W., J. Chromatogr., 559:401 (1991).

[0224] 42. Satow, T. et al., “The effecto of salts on the separation ofbioactive peptides by capillary electrophoresis,” J. High Resolut.Chromatogr., 14:276 (1991).

[0225] 43. Shihabi, Z. K. and L. L. Garcia, “Effects of sample matrix onseparation by capillary electrophoresis,” in: CRC Handbook of CapillaryElectrophoresis: A Practical Approach, Chp. 20, pgs. 537-548 (CRC Press,Boca Raton, Fla., 1994).

[0226] 44. Garfin, D. E., Methods in Enzymology, 182:425 (1990).

[0227] 45. Jorgenson, J. W. and K. D. Lukacs, “Zone electrophoresis inopen-tubular glass capillaries: preliminary data on performance,” J.High Resolut. Chromatogr. Commun., 4:230 (1981).

[0228] 46. Jorgenson, J. W., and K. D. Lukacs, “Zone electrophoresis inopen tubular capillaries,” Anal. Chem., 53:1298 (1981).

[0229] 47. Mc Cormick, R. M., “Capillary zone electrophoresis ofpeptides,” in: CRC Handbook of Capillary Electrophoresis: A PracticalApproach, Chp. 12, pgs. 287-323 (CRC Press, Boca Raton, Fla., 1994).

[0230] 48. Aebersold, R., “Proteome analysis: Biological assay or dataarchive?,” paper presented at the IBC Proteomics conference, Coronado,Calif. (Jun. 11-12, 1998).

[0231] 49. Cobb, K. A. and M. Novotny, “High-sensitivity peptide mappingby capillary zone electrophoresis and microcolumn liquid chromatography,using immobilized trypsin for protein digestion, Anal. Chem., 61:2226(1989).

[0232] 50. Cantor, C. R. and P. R. Schimmel, Biophysical Chemistry (W.H.Freeman & Co., NY, 1980).

[0233] 51. Hjerten, S., “Free zone electrophoresis,” Chromatogr. Rev.,9:122 (1967).

[0234] 52. Martinek, K., Goldmacher, V. S., Klibanov, A. M., andBerezin, I. V., “Denaturing agents (urea, acrylamide) protect enzymesagainst irreverisble thermoinactivation: A study with native andimmobilized alpha-chymotrypsin and trypsin,” FEBS Lett., 51:152-155(1975).

[0235] 53. Altria, K. D. and C. F. Simpson, “Measurement ofelectroendosmosis in high-voltage capillary electrophoresis,” Anal.Proc., 23:453 (1986).

[0236] 54. Camilleri, P. and G. N. Okafo, “Replacement of H₂O by D20 incapillary zone electrophoresis can increase resolution of peptides andproteins, “J. Chem. Soc. Chem. Commun., 3:196 (1991).

[0237] 55. Camilleri, P., G. N. Okafo, C. Southan, and R. Brown,“Analytical and micropreparative capillary electrophoresis of thepeptides from calcitonin,” Anal. Biochem., 198:36 (1991).

[0238] 56. Okafo, G. N. and P. Camilleri, “Capillary electrophoreticseparation in both H₂O and [2H]2O-based electrolytes can provide moreinformation on tryptic digests, J. Chromatogr., 547:551 (1991).

[0239] 57. Schwer, C. and F. Lottspeich, “Analytical andmicropreparative separation of peptides by capillary zoneelectrophoresis using discontinuous buffer systems,” J. Chromatogr.,623:345 (1992).

[0240] 58. Foret, F., E. Szoko and B. L. Karger, “On-column transientand coupled column isotachophoretic preconcentration of protein samplesin capillary zone electrophoresis,” J. Chromatogr., 608:3 (1992).

[0241] 59. Lowry, O., N. Rosebrough, A. Farr and R. Randall, J. Biol.Chem., 193:265-275 (1951).

[0242] 60. Anderson, N. L., “Pharmaceutical Proteomics: Targets,mechanism, and function,” paper presented at the IBC Proteomicsconference, Coronado, Calif. (Jun. 11-12, 1998).

[0243] 61. Meng, C. K., M. Mann and J. B. Fenn, Z. Phy. D: Atoms, Mol.Clusters, 10:361-368 (1988).

[0244] 62. Karas, M. and F. Hillenkamp, Anal. Chem. 60:2299 (1988).

[0245] 63. Hillenkamp, F., M. Jaras, R. C. Beavis and B. T. Chait, Anal.Chem. 63:1193A (1991).

[0246] 64. Beavis, R. C. and B. T. Chait, “Matrix-assisted laserdesorption mass spectrometry of proteins,” preprint,http://www.proteometrics.com/methods/contents.htm (1994).

[0247] 65. Clauser, K. R., S. C. Hall, D. M. Smith, J. W. Webb, L. E.Andrews, H. M. Tran, L. B. Epstein, and A. L. Burlingame, Proc. Natl.Acad. Sci (USA), 92:5072-5076 (1995).

[0248] 66. Li, G., M. Walthan, N. L. Anderson, E. Unworth, A. Trestonand J. N. Weinstein, “Rapid mass spectrometric identification ofproteins from two-dimensional polyacrylamide gels after in gelproteolytic digestion,” Electrophoresis, 18:391-402 (1997).

[0249] 67. Stevens, F. J., “Method of electric field flow fractionationwherein the polarity of the electric field is periodically reversed,”U.S. Pat. No. 5,133,844, (Jul. 28, 1992).

[0250] 68. Gupta N. R., Nadim A., Haj-Hariri H., Borhan A., “Stabilityof the Shape of a Viscous Drop under Buoyancy-Driven Translation in aHele-Shaw Cell, “J Colloid Interface Sci, 222(1):107-116 (2000).

[0251] 69. Sanger, F. Biochem. J., 39:507 (1945).

[0252] 70. Creighton, T. E., Proteins: Structures and MolecularPrinciples (W. H. Freeman, NY, 1984).

[0253] 71. Niederwieser, A., “Thin-layer chromatography of amino acidsand derivatives,” in: Methods in Enzymology, 25:60-99 (1972).

[0254] 72. Hirs, C. H. W., M. Halmann and J. H. Kycia,”Dinitrophenylation and inactivation of bovine pancreatic ribonucleaseA,” Arch. Biochem. Biophys., 111:209-222 (1965).

[0255] 73. Gray, W. R., “End-group analysis using dansyl chloride,” in:Methods in Enzymology, 25:121-137 (1972).

[0256] 74. Stark, G. R., “Use of cyanate for determining NH2-terminalresidues in protein,” in: Methods in Enzymology, 25:103-120 (1972).

[0257] 75. Niall, H. D., “Automated Edman degradation: the proteinsequenator,” in: Methods in Enzymology, 27:942-1011 (1973).

[0258] 76. Galella, G. and D. B. Smith, “The cross-linking of tubulinwith immidoesters,” Can. J. Biochem., 60:71-80 (1982).

[0259] 77. Lomant, A. J. and G. Fairbanks, “Chemical probes of extendedbiological structures: synthesis and properties of the cleavable proteincrosslinking reagent 35S dithiobis(succinimidyl propionate), J. Mol.Biol., 104:243-261 (1976).

[0260] 78. Solomons, T. W. G, Organic Chemistry (John Wiley & Sons, NY,1976).

[0261] 79. Novotny et al., Anal. Chem., 63:408 (1991).

[0262] 80. Novotny et al., J. Chromatography, 499:579 (1990).

[0263] 81. Merrifield, B., Science, 232:341-347 (1986).

[0264] 82. Horton, H. R. and D. E. Koshland, Jr., Methods in Enzymology,25:468 (1972).

[0265] 83. Yamada, H., Imoto, T., Fujita, K., Okazaki, K. and M.Motomura, “Selective modification of aspartic acid-101 in lysozyme bycarbodiimide reaction,” Biochem., 20:4836-4842.

[0266] 84. Grabarek, Z. and J. Gergely, “Zero-length crosslinkingprocedure with the use of active esters,” Anal. Biochem. 185:131-135(1990).

1-61. (Cancelled)
 62. A method for separating a plurality of proteins,comprising performing a capillary isoelectric focusing electrophoresis(CIEF) or capillary zone electrophoresis (CZE) method with a samplecontaining the plurality of proteins, wherein the CIEF method or CZEmethod is conducted under conditions such that electroosmotic flow (EOF)is less than or equal to 0.5×10-6 cm²/V-s.
 63. The method of claim 62,wherein the method comprises performing a CIEF method under conditionssuch that electroosmotic flow (EOF) is less than or equal to 0.5×10⁻⁶cm²/V-s.
 64. The method of claim 62, wherein the method comprisesperforming a CZE method under conditions such that electroosmotic flow(EOF) is less than or equal to 0.5×10⁻⁶ cm²/V-s.
 65. The method of claim62, further comprising detecting at least one protein separated by theCIEF or CZE method.
 66. The method of claim 65, further comprisinganalyzing the at least one protein to determine a chemical or physicalcharacteristic of the at least one protein.
 67. The method of claim 66,wherein the chemical or physical characteristic is selected from thegroup consisting of molecular weight, complete or partial amino acidsequence, isoelectric point, relative or absolute abundance andcombinations of the foregoing.
 68. The method of claim 66, whereinanalyzing is conducted by mass spectrometry.